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.     

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 relationships of two pairs of closely related O-antigens of Escherichia coli and Salmonella enterica: E. coli O11/S. enterica O16 and E. coli O21/S. enterica O38

The O-antigen is a part of the lipopolysaccharide molecule present in the outer membrane of Gram-negative bacteria, and is essential for the full function of the microorganisms. Salmonella enterica and Escherichia coli are taxonomically closely related species. In this study, the O-antigen structures of S. enterica O16 and O38 and E. coli O11 were determined. Salmonella enterica O38 and E. coli O21 were found to have identical O-antigen structures, whereas S. enterica O16 and E. coli O11 had closely related structures, differing only in the presence of a lateral glucose residue and O-acetylation of a mannose residue in the former. The O-antigen gene clusters of S. enterica O16 and O38 and E. coli O11 were sequenced and analyzed together with that of E. coli O21 retrieved from the GenBank. Each S. enterica/E. coli pair was found to contain the same set of genes organized in the same manner and to share 56-78% overall DNA identity. These data suggest that the O-antigen gene clusters of each pair studied originated from a common ancestor. Thus, it has become evident that in the past, the degree of relatedness between the O-antigens of S. enterica and E. coli was underestimated.     

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.     

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.     

The colanic acid gene cluster of Salmonella enterica has a complex history

The colanic acid gene cluster of Salmonella enterica LT2 was sequenced and compared with that of Escherichia coli K-12. The two clusters are similar with divergence slightly higher than average for genes of the two species. The cluster was divided into four blocks by GC content and seems likely to have transferred from a higher GC content species to the ancestor of E. coli and S. enterica. All 19 genes of K-12 and 13 genes of LT2 appear to have undergone random genetic drift with amelioration of the GC content. However, in the case of S. enterica, we believe that the six genes of the GDP-fucose pathway group were replaced relatively recently by genes closely related to those of the original donor species. Two repetitive elements were observed: a bacterial interspersed mosaic element in the intergenic region between wzx and wcaK in K-12 only and a RSA (repetitive sequence element) sequence between wcaJ and wzx in LT2 only.     

Murine monoclonal antibodies specific for lipopolysaccharide of Escherichia coli O26 and O111

Monoclonal antibody (MAb) 12F5 reacted with 35 Escherichia coli O26 isolates and cross-reacted with 1 of 365 non-E. coli O26 isolates. MAb 15C4 reacted with 30 E. coli O111 strains and 8 Salmonella O35 strains (possessing identical O antigen) but not with 362 other bacterial strains. Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.     

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.     

The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene

Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.     

Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1).

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.     

Evolutionary origins and sequence of the Escherichia coli O4 O-antigen gene cluster

Escherichia coli express many types of O antigen, present in the outer membrane of the Gram-negative bacterial cell wall. O-Antigen biosynthesis genes are clustered together and differences seen in O-antigen types are due to genetic variation within this gene cluster. Sequencing of the E. coli O4 O-antigen gene cluster revealed a similar gene order and high levels of similarity to that of E. coli O26; indicating a common ancestor. These lateral transfer events observed within O-antigen gene clusters may occur as part of the evolution of the pathogenic clones.     

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.     

Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assay

AIM: To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. METHODS AND RESULTS: DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg microl(-1) chromosomal DNA, 0.2 CFU g(-1) pork and 0.2 CFU ml(-1) water. The total time required from beginning to end of the procedure was within 16 h. CONCLUSION: The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens.     

Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci.     

Two functional O-polysaccharide polymerase wzy (rfc) genes are present in the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum

Defined regions of the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum were introduced into a mutated derivative of this strain that lacks O-polysaccharide polymerase activity. Three different kinds of assays performed on the various transformants all indicate that two functional wzy (rfc) genes reside within the Group E1 Salmonella rfb gene cluster. The product of ORF9.6, positioned near the center of the rfb gene cluster, joins O-polysaccharide repeat units together by alpha-glycosidic linkages to produce antigen O10, the major serological determinant of Group E1 S. enterica. The product of ORF17.4, positioned at the downstream end of the rfb gene cluster, can join repeat units together by beta-glycosidic linkages to produce antigen O15, the major serological determinant of Group E2 S. enterica.     

Development of PCR assays targeting genes in O-antigen gene clusters for detection and identification of Escherichia coli O45 and O55 serogroups

The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 10(6) and 10(8) CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.     

Detection of Escherichia coli serogroup O103 by real-time polymerase chain reaction

AIMS: The aims of the study were to identify the specific genes of O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O103 and to provide the basis for a specific real-time PCR test for rapid detection of E. coli O103. 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 12-kbp O103 O-antigen biosynthesis locus of STEC O103. A DNA library representative of this cluster allowed two O103-specific probes to be identified in the flippase (wzx) and UDP-galactose-4-epimerase (galE) genes. Two specific O103 serotyping real-time PCR tests based on these two genes were successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen real-time PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of real-time PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.     

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.     

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.

The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).