Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIA.

Salmonella enterica and Yersinia pseudotuberculosis are the only examples in nature known to use a variety of 3,6-dideoxyhexose derivatives as O antigen constituents. To allow a comparison of the responsible biosynthetic genes of the two organisms, we have sequenced a section of the Y. pseudotuberculosis serogroup IIA rfb region that contained the genes for the abequose biosynthetic pathway. Comparison of the identified genes with the rfb region of S. enterica LT2 showed that the two dideoxyhexose pathway gene clusters are related. The arrangement of the genes was largely conserved, and the G + C compositions of the two DNA regions were strikingly similar; however, the degree of conservation of nucleotide and protein sequences suggested that the two gene clusters have been evolving independently for considerable time. Hybridization experiments showed that the dideoxyhexose pathway genes are widespread throughout the various serogroups of Y. pseudotuberculosis.     

Identification of an unusual [2Fe-2S]-binding motif in the CDP-6-deoxy-D-glycero-l-threo-4-hexulose-3-dehydrase from Yersinia pseudotuberculosis: implication for C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses

CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E(1)) catalyzes the C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. E(1) is a pyridoxamine 5'-phosphate (PMP)-dependent enzyme that also contains a [2Fe-2S] center. This iron-sulfur cluster is catalytically essential, since removal of the [2Fe-2S] center leads to inactive enzyme. To identify the [2Fe-2S] core in E(1) and to study the effect of impairing the iron-sulfur cluster on the activity of E(1), a series of E(1) cysteine mutants were constructed and their catalytic properties were characterized. Our results show that E(1) displays a cluster-binding motif (C-X(57)-C-X(1)-C-X(7)-C) that has not been observed previously for [2Fe-2S] proteins. The presence of such an unusual iron-sulfur cluster in E(1), along with the replacement of the active site lysine by a histidine residue (H220), reflects a distinct evolutionary path for this enzyme. The cysteine residues (C193, C251, C253, C261) implicated in the binding of the iron-sulfur cluster in E(1) are conserved in the sequences of its homologues. It is likely that E(1) and its homologues constitute a new subclass in the family of iron-sulfur proteins, which are distinguished not only by their cluster ligation patterns but also by the chemistry used in catalyzing a simple, albeit mechanistically challenging, reaction.     

Structure and mutagenic conversion of E1 dehydrase: at the crossroads of dehydration, amino transfer, and epimerization

Pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are highly versatile coenzymes whose importance is well recognized. The capability of PLP/PMP-dependent enzymes to catalyze a diverse array of chemical reactions is attributed to fine-tuning of the cofactor-substrate interactions in the active site. CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1), along with its reductase (E3), catalyzes the C-3 deoxygenation of CDP-4-keto-6-deoxy-D-glucose to form the dehydrated product, CDP-4-keto-3,6-dideoxy- d-glucose, in the ascarylose biosynthetic pathway. This product is the progenitor to most 3,6-dideoxyhexoses, which are the major antigenic determinants of many Gram-negative pathogens. The dimeric [2Fe-2S] protein, E 1, cloned from Yersinia pseudotuberculosis, is the only known enzyme whose catalysis involves the direct participation of PMP in one-electron redox chemistry. E1 also contains an unusual [2Fe-2S] cluster with a previously unknown binding motif (C-X 57-C-X 1-C-X 7-C). Herein we report the first X-ray crystal structure of E1, which exhibits an aspartate aminotransferase (AAT) fold. A comparison of the E1 active site architecture with homologous structures uncovers residues critical for the dehydration versus transamination activity. Site-directed mutagenesis of four E1 residues, D194H, Y217H, H220K, and F345H, converted E 1 from a PMP-dependent dehydrase to a PLP/glutamate-dependent aminotransferase. The E1 quadruple mutant, having been conferred this altered enzyme activity, can transaminate the natural substrate to CDP-4,6-dideoxy-4-amino-D-galactose without E3. Taken together, these results provide the molecular basis of the functional switch of E1 toward dehydration, epimerization, and transamination. The insights gained from these studies can be used for the development of inhibitors of disease-relevant PLP/PMP-dependent enzymes.     

A retro-evolution study of CDP-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase (E1) from Yersinia pseudotuberculosis: implications for C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses

CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B6-dependent enzymes, albeit with two important distinctions. It is a rare example of a B6-dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B6-dependent enzymes is replaced by histidine at position 220 in E1. Since alteration of His220 to a lysine residue may produce a putative progenitor of E1, the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic mechanism of E1.     

CDP-6-deoxy-delta 3,4-glucoseen reductase from Yersinia pseudotuberculosis: enzyme purification and characterization of the cloned gene.

The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol I) including a trypsin digestion step (O. Han, V.P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E3 activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E3. A reinvestigation of the purification protocol produced a new and improved procedure (protocol II) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascD) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E3. The nucleotide and translated amino acid sequences of the genuine E3 are also presented.     

Mechanistic and stereochemical studies of a unique dehydration catalyzed by CDP-4-keto-6-deoxy-D-glucose-3-dehydrase: a pyridoxamine 5'-phosphate dependent enzyme isolated from Yersinia pseudotuberculosis.

CDP-4-keto-6-deoxy-D-glucose-3-dehydrase (E1) purified from Yersinia pseudotuberculosis is a pyridoxamine 5'-phosphate (PMP) dependent enzyme which catalyzes the C-O bond cleavage at C-3 of a CDP-4-keto-6-deoxy-D-glucose substrate, a key step in the formation of 3,6-dideoxyhexoses. Since enzyme E1 utilizes the PMP cofactor in a unique manner, it is essential to establish its role in E1 catalysis. When an incubation was conducted in [18O]H2O, incorporation of 18O into positions C-3 and C-4 of the recovered substrate was observed. This result not only provided the evidence necessary to reveal the reversibility of E1 catalysis but also lent credence to the formation of a delta 3,4-glucoseen intermediate. In view of E1 catalysis being initiated by a C-4' deprotonation of the PMP-substrate complex, the stereochemical course of this step was examined using chemically synthesized (4'S)- and (4'R)-[4'-3H]PMP as probes. Our results clearly demonstrated that the stereochemistry of this deprotonation is pro-S specific, which is in agreement with the stereochemical consistency found with other vitamin B6 phosphate dependent enzymes. The fact that reprotonation at C-4' of the PMP-delta 3,4-glucoseen complex in the reverse direction of E1 catalysis was also found to be pro-S stereospecific strongly suggested that enzyme E1, like most of its counterparts, has the si face of its cofactor-substrate complex exposed to solvent and accessible to active-site catalytic groups as well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of O-specific side chains of lipopolysaccharides from Yersinia pseudotuberculosis

Lipopolysaccharide prepared from cells of Yersinia (Pasteurella) pseudotuberculosis of serogroups I, II, III, IV, and V is known to contain the 3,6-dideoxyhexose (DDH) paratose, abequose, paratose, tyvelose, and ascarylose in its respective O-specific side chains. Lipopolysaccharides or lipid-free polysaccharides of all of the 10 known serogroups and subgroups were subjected to methylation analysis and determined as alditol acetates by gas-liquid chromatography and mass spectrometry. The results indicated that the O-specific side chains of nine serotypes are composed of oligosaccharide repeating units in the form of four alternative general structures in which a terminal DDH may vary. These structures are DDH [Formula: see text] 6-deoxy-d-manno-heptose [Formula: see text] d-galactose (serogroups IA, IIA, and IVB), DDH [Formula: see text] d-mannose [Formula: see text] l-fucose (serogroups IB and IIB), and two configurations similar to the latter except that the 4-position of l-fucose was either linked to the d-mannose residue (serogroups VA and VB) or to the DDH residue (serogroups III and IVA). In contrast, O-groups in lipopolysaccharide of the newly discovered serogroup VI contained the DDH colitose and 2-acetamido-2-deoxy-d-galactose. Accordingly, all five known types of DDH have now been detected in lipopolysaccharides of Y. pseudotuberculosis. The sugar 6-deoxy-d-manno-heptose, present in O-specific side chains of serogroups IA, IIA, and IVB, has not yet been reported to occur elsewhere in nature.     

Synthesis of cytidine diphosphate-3,6-dideoxyhexoses--donors of glycosyl residues in the biosynthesis of O-specific polysaccharides of Salmonella of serogroups A, B and C

Interaction of lithium alcoholates of 2,4-di-O-benzoates of paratose and abequose with tetrabenzyl pyrophosphate gave alpha-phosphates of the 3,6-dideoxyhexoses, further converted into the corresponding cytidine-5'-diphosphate derivatives. These synthetic nucleotides were shown to participate in the biosynthesis of the O-specific polysaccharides for Salmonella typhimurium and S. nitra.     

Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis: purification and characterization of CDP-4-keto-6-deoxy-D-glucose-3-dehydrase.

CDP-4-keto-6-deoxy-D-glucose-3-dehydrase (E1) is a PMP-dependent enzyme which plays an essential role in C-O bond cleavage leading to the formation of 3,6-dideoxyhexoses. Although E1 catalysis has long been recognized as a unique biological deoxygenation reaction, the catalytic mechanism of this unusual enzyme has never been fully elucidated. The lack of methods that would allow this enzyme's activity to be monitored directly has been an impediment to E1 purification and has consequently hampered the mechanistic studies. In order to circumvent this problem, we have developed a few convenient and sensitive methods to facilitate the E1 assay. The first method relies on the fact that E1-catalyzed dehydration is initiated by a proton abstraction from C-4' of the PMP-substrate adduct. By using a tritium-labeled cofactor in the incubation that was later quenched with charcoal, the amount of E1 present could be determined from the amount of released tritium in the supernatant. The second method was designed on the basis of the expectation that E1 will bind and rupture the C-F bond of a substrate analogue, CDP-4-keto-3,6-dideoxy-3-fluoro-D-glucose, which was derived from CDP-3-deoxy-3-fluoro-D-glucose. Since the bond length and electronegativity of the C-F group are similar to those of a C-OH group, we anticipated that the proposed compound would be processed by E1, an assumption which was later substantiated. Another assay useful for measuring E1 activity couples the E1 transformation with the subsequent reduction step catalyzed by CDP-6-deoxy-delta 3,4-D-glucoseen reductase (E3) to a thiobarbituric acid (TBA) reaction. Since the condensation product of TBA and malonaldehyde derived from oxidative degradation of the E1/E3 product gave a pink chromophore at 532 nm with a known absorption coefficient, the yield of deoxysugar formation could be directly deduced on the basis of the observed absorbance. The most conclusive evidence confirming the role of E1 was attained by a GC/MS assay which permits an unambiguous identification of the deoxysugar product generated from the E1 and E3 reactions. With these convenient and sensitive assays in hand, we have established a sequence of four columns that was effective in consistently producing pure E1 from Yersinia pseudotuberculosis. The overall purification may be as high as 26,000-fold. This purified enzyme consists of a single polypeptide chain in its native form, and the estimated molecular weight is 49,000.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic analysis of the O-antigen gene clusters of Yersinia pseudotuberculosis O:6 and O:7

Among the 21 O-polysaccharide (OPS) O-antigen-based serotypes described for Yersinia pseudotuberculosis, those of O:6 and O:7 are unusual in that both contain colitose (4-keto-3,6-dideoxy-d-mannose or 4-keto-3,6-dideoxy-l-xylo-hexose), which has not otherwise been reported for this species, and the O:6 OPS also contains yersiniose A (4-C[(R)-1-hydroxyethyl]-3,6-dideoxy-d-xylo-hexose), another unusual dideoxyhexose sugar. In Y. pseudotuberculosis, the genes for OPS synthesis generally cluster together between the hemH and gsk loci. Here, we present the sequences of the OPS gene clusters of Y. pseudotuberculosis O:6 and O:7, and the location of the genes required for synthesis of these OPSs, except that there is still ambiguity regarding allocation of some of the glycosyltransferase functions. The O:6 and O:7 gene clusters have much in common with each other, but differ substantially from the group of 13 gene clusters already sequenced, which share several features and sequence similarities. We also present a possible sequence of events for the derivation of the O:6 and O:7 gene clusters from the most closely related set of 13 sequenced previously.     

Modeling of deoxy- and dideoxyaldohexopyranosyl ring puckering with MM3(92)

Extensive variations of the ring structures of three deoxyaldohexopyranoses, L-fucose, D-quinovose, and L-rhamnose, and four dideoxyaldohexopyranoses, D-digitoxose, abequose, paratose, and tyvelose, were studied by energy minimization with the molecular mechanics algorithm MM3(92). Chair conformers, 4C(1) in D-quinovose and the equivalent 1C(4) in L-fucose and L-rhamnose, overwhelmingly dominate in the three deoxyhexoses; in the D-dideoxyhexoses, 4C(1) is again dominant, but with increased amounts of 1C(4) forms in the alpha anomers of the three 3,6-dideoxyhexoses, abequose, paratose, and tyvelose and in both alpha and beta anomers of the 2,6-dideoxyhexose D-digitoxose. In general, modeled proton-proton coupling constants agreed well with experimental values. Computed anomeric ratios strongly favor the beta configuration except for D-digitoxose, which is almost equally divided between alpha and beta configurations, and L-rhamnose, where the beta configuration is somewhat favored. MM3(92) appears to overstate the prevalence of the equatorial beta anomer in all three deoxyhexoses, as earlier found with fully oxygenated aldohexopyranoses.     

The genetics and structure of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:10 and its relationship with Escherichia coli O111 and Salmonella enterica O35

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

Biosynthesis of colitose: expression, purification, and mechanistic characterization of GDP-4-keto-6-deoxy-D-mannose-3-dehydrase (ColD) and GDP-L-colitose synthase (ColC)

L-Colitose is a 3,6-dideoxyhexose found in the O-antigen of Gram-negative lipopolysaccharides. To study the biosynthesis of this unusual sugar, we have cloned and sequenced the L-colitose biosynthetic gene cluster from Yersinia pseudotuberculosis VI. The colD and colC genes in this cluster have been overexpressed and each gene product has been purified and characterized. Our results showed that ColD functions as GDP-4-keto-6-deoxy-D-mannose-3-dehydrase responsible for C-3 deoxygenation of GDP-4-keto-6-deoxy-D-mannose. This enzyme is coenzyme B(6)-dependent and its catalysis is initiated by a transamination step in which pyridoxal 5'-phosphate (PLP) is converted to pyridoxamine 5'-phosphate (PMP) in the presene of L-glutamate. This coenzyme forms a Schiff base with the keto sugar substrate and the resulting adduct undergoes a PMP-mediated beta-dehydration reaction to give a sugar enamine intermediate, which after tautomerization and hydrolysis to release ammonia yields GDP-4-keto-3,6-dideoxy-D-mannose as the product. The combined transamination-deoxygenation activity places ColD in a class by itself. Our studies also established ColC as GDP-L-colitose synthase, which is a bifunctional enzyme catalyzing the C-5 epimerization of GDP-4-keto-3,6-dideoxy-D-mannose and the subsequent C-4 keto reduction of the resulting L-epimer to give GDP-L-colitose. Reported herein are the detailed accounts of the overexpression, purification, and characterization of ColD and ColC. Our studies show that their modes of action in the biosynthesis of GDP-L-colitose represent a new deoxygenation paradigm in deoxysugar biosynthesis.     

Incorporation of paratose residue into Salmonella O-specific polysaccharides using enzymatic reactions]

The block mechanism of O-specific polysaccharides biosynthesis was demonstrated for Salmonella nitra (serogroup A) and S. haifa (serogroup B). Due to the moderate specificity of glycosyl transferases from S. nitra, S. typhimurium, S. haifa and S. kentucky (serogroup C3) towards the 3,6-dideoxyhexose structure a paratose residue can be incorporated into the polysaccharide chain instead of an abequose residue, and vice versa.     

The Yersinia high-pathogenicity island.

A pathogenicity island present only in highly pathogenic strains of Yersinia (Y. enterocolitica 1B, Y. pseudotuberculosis I and Y. pestis) has been identified on the chromosome of Yersinia spp. and has been designated High-Pathogenicity Island (HPI). The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin. The major function of this island is thus to acquire iron molecules essential for in vivo bacterial growth and dissemination. The presence of an integrase gene and att sites homologous to those of phage P4, together with a G + C content much higher than the chromosomal background, suggests that the HPI is of foreign origin and has been acquired by chromosomal integration of a phage. The HPI can excise from the chromosome of Y. pseudotuberculosis and is found inserted into any of the three copies of the asn tRNA loci present in this species. A unique characteristic of the HPI is its wide distribution in various enterobacteria. Although first identified in Yersinia spp., it has subsequently been detected in other genera such as E. coli, Klebsiella and Citrobacter.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Complete physical map of the rfb gene cluster encoding biosynthetic enzymes for the O antigen of Salmonella typhimurium LT2.

Biosynthesis of the Salmonella typhimurium LT2 O antigen is encoded by genes which map in the rfb cluster. The cloning and restriction enzyme analysis of part of this cluster have been described previously (H. N. Brahmbhatt, N. B. Quigley, and P. R. Reeves, Mol. Gen. Genet. 203:172-176, 1986). The entire rfb gene cluster has now been cloned, and a detailed restriction enzyme map has been constructed which has enabled us to map the approximate positions of individual rfb genes.     

Genetic Transfer of Salmonella typhimurium and Escherichia coli Lipopolysaccharide Antigens to Escherichia coli K-12

Escherichia coli K-12 varkappa971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv(+) hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his(+) (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F' factor (FS400) carrying the rfb-his region of S. typhimurium to the same two ilv(+) hybrids gave similar results. LPS extracted from two ilv(+),his(+), factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his(+) hybrids obtained from varkappa971 itself by similar HfrK9 and F'FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli varkappa971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli varkappa971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli varkappa971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his(+) recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Omega8. This suggests that, although the parental E. coli K-12 strain varkappa971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units.     

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.