Antigen Gene Cluster Research Articles

Pathogenesis of IgA nephropathy

Immunoglobulin A (IgA) nephropathy is an immune-complex-mediated glomerulonephritis characterized by the presence of immunoglobulin A deposits in mesangial and paramesangial regions. The patients with IgA nephropathy present with varying clinical symptoms (eg, microhematuria with preserved renal function or progressive deterioration of renal functions resulting in end-stage renal disease). The factors involved in the pathogenetic mechanisms of IgA nephropathy include (1) environmental factors, (2) genetic factors, (3) abnormality of the IgA1 molecule, and (4) various inflammatory mediators. The gene polymorphism studies for human leukocyte antigen (HLA), renin-angiotensin-aldosterone system, and selectin gene clusters, suggest a certain degree of genetic predisposition in patients for IgA nephropathy. Also, the genome-wide screening in familial IgA nephropathy showed linkage of IgA nephropathy to the 6q22–23 chromosome. Besides genetic influence in its pathogenesis, aberrant galactosylation in serum IgA and IgA1 eluted from kidneys with IgA nephropathy has been observed, and conceivably such abnormalities induce the expression of various cytokines, interleukin (IL)-6, platelet-derived growth factor (PDGF), tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 in the renal cells, which contributes to further glomerular injury. Despite an enormous amount of information available in the literature, further studies are needed to delineate the precise pathogenetic mechanisms involved in primary IgA nephropathy and also to facilitate the development of newer therapeutic interventions.

The variation of dTDP-L-rhamnose pathway genes in Vibrio cholerae.

The genetic variation in the dTDP-L-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated. The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters. The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes. Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes. Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5' end of the gene clusters. A gradient in the level of variation was observed, with highly similar sequences at the 5' end rmlB gene, but very divergent and strain-specific sequences at the 3' end of the rml gene set. The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5' and 3' parts are of different origin, and that recombination within rml genes has occurred. The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced. Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets. This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V. cholerae. The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V. cholerae and DNA that probably came into the species with the O antigen gene cluster.

Increased adherence to Caco-2 cells caused by disruption of the yhiE and yhiF genes in enterohemorrhagic Escherichia coli O157:H7.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelium is essential for initiation of infections, including diarrhea, and expression of the genes of the locus of enterocyte effacement (LEE) is thought to be crucial for adherence. To identify genes involved in modulating the adherent capacity, bacteria collected from an EHEC O157:H7 strain (O157Sakai) mutagenized by mini-Tn5Km2 were screened for their ability to increase the number of microcolonies (MC) on Caco-2 cells and eight mutants with increased adherence were isolated. Analysis of the mini-Tn5Km2-flanked DNA sequences indicated that one possessed the insertion within an O157 antigen gene cluster, another possessed the insertion within the yhiF gene, and the remaining six mutants had their insertions in the yhiE gene. yhiE and yhiF products share amino acid homology (23% identity) to each other and with members of the LuxR family, which are known as transcriptional regulatory proteins. The mutant having the insertion within the O157 antigen gene cluster did not express the O157 side chain (as determined by agglutination test and immunoblotting with polyclonal O157-specific antiserum), unlike the other seven mutants. Importantly, the other mutants showed enhanced type III secretion. Levels of the related mRNAs of genes of the LEE, but not that of ler mRNA, were also increased compared with those in the wild type. Indeed, when we introduced an in-frame deletion into the yhiE or yhiF gene in O157Sakai, the capacity of the resultant mutants to adhere to Caco-2 cells was greatly increased. When one of the yhiE insertion mutants was orally inoculated into ICR mice, the number of bacteria shed into feces by day 14 was greater than that for the wild type. These results suggest that yhiE and yhiF are involved in the adherence of O157Sakai to epithelial cells as negative regulators for the expression of the genes required for the type III secretion system.

Sequence of the Escherichiacoli O26 O antigen gene cluster and identification of O26 specific genes

Escherichia coli associated with outbreaks of gastroenteritis and hemolytic uremic syndrome include clones with O antigens O157 and O111. However, O26 has emerged as an O antigen present in pathogenic strains, particularly those implicated in cases of infantile gastroenteritis worldwide. The O26 O antigen gene cluster was sequenced. It was found to contain the genes expected for biosynthesis of nucleotide sugars l-rhamnose, N-acetyl- l-fucosamine and N-acetyl-glucosamine, as well genes for O unit flippase, O antigen polymerase and potential transferase genes. By polymerase chain reaction testing against representative strains for the 166 Escherichia coli O serogroups and some randomly selected Gram-negative bacteria, we identified three O antigen genes that are highly specific to O26. This work provides the basis for a sensitive test for the rapid detection of pathogenic clones with the O26 antigen, which has implications for public health, especially in the control of food-borne outbreaks.

Evidence that the enterococcal polysaccharide antigen gene (epa) cluster is widespread in Enterococcus faecalis and influences resistance to phagocytic killing of E. faecalis.

In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (epa) from Enterococcus faecalis strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in Escherichia coli. Disruption of two open reading frames in the epa gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with E. faecalis endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (P < or = 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (P < or = 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the epa gene cluster of OG1RF (which is also present in the sequenced E. faecalis strain V583) was detected in 12 of 12 other clonally distinct E. faecalis strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an E. faecalis endocarditis patient serum, and sera from four other patients with E. faecalis endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the epa gene cluster is widespread among E. faecalis and confers some protection against human host defenses.

Sequence of the E.coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.

Molecular evolution of the GDP-mannose pathway genes (manB and manC) in Salmonella enterica.

The evolutionary history of the GDP-mannose pathway in Salmonella enterica was studied via sequencing manB and manC genes from 13 representative strains for O antigens containing mannose and/or sugar derivatives of GDP-D-mannose. In addition, colanic acid (CA) manB and manC genes were sequenced from selected strains, as the basis for a detailed comparison. Interestingly, including the eight previously characterized O antigen gene clusters, 12 of the 21 S. enterica strains studied in total (each representing a different O antigen structure) possess a manB gene which displays DNA identity, ranging from 93 to 99%, to the CA manB gene of S. enterica LT2. Furthermore, the CA-like manB genes (as well as the CA manB and manC genes) display subspecies specificity, and the CA and CA-like manB genes (for individual strains) appear to be evolving in concert via gene conversion events. In comparison, the manC genes were generally not CA-like, a situation also apparent in Escherichia coli,and therefore most strongly reflected the evolutionary history of the S. enterica O antigen GDP-mannose pathway. It appears that, in relatively recent times, gene capture from a distant source has occurred infrequently, and that groups of manB and manC genes have been maintained and are continuing to evolve within S. enterica and more closely related species.

Genetic variation of dTDP-L-rhamnose pathway genes in Salmonella enterica.

The genetic variation in the dTDP-L-rhamnose pathway gene set (rmlB, rmlD, rmlA, rmlC) in Salmonella enterica was examined after sequencing the four genes from 11 rml-containing gene clusters encoding seven O antigens, and a 903 bp rmlB segment from another 23 strains representing the seven subspecies. There was considerable sequence variation and strong polarity in the nature and level of variation among rml genes. The 5' end of the rml gene set, including rmlB, rmlD and most of rmlA, is in general subspecies specific. In contrast, the 3' end, including part of rmlA and all of rmlC, is O antigen specific. The G+C content of the 3' end is lower than that of the 5' end. The variation in the 3' end of the gene set is much greater than that of the 5' end. It is apparent that the rml gene set of S. enterica includes genes with two different evolutionary histories. In addition, there has been extensive recombination in the gene set, probably related to O antigen transfer between subspecies. These findings provide evidence for the lateral transfer of O antigen genes between species and among subspecies of S. enterica. The results have also shown that conserved genes at the end of an O antigen gene cluster play a major role in mediating exchange of the central serogroup-specific regions.

Genetic basis of psoriasis

Psoriasis manifests between the ages of 20 and 40 and is characterized by plaque-like hyperproliferative skin lesions, particularly at sites of trauma. It affects around 1–2% of the population and can be associated with other inflammatory diseases. Disease can be triggered by climate, stress and, of course, genetic factors. Like many other complex genetic diseases, no specific susceptibility mutations have been identified as yet, although linkage to a number chromosome regions has been demonstrated, such as 17q25. The strongest genetic association for psoriasis is with the human leukocyte antigen (HLA) gene cluster on 6p21, and specifically linkage with the HLA-Cw6 allele. Genomic sequence of the chromosome region harbouring the psoriasis-susceptibility locus PSORS1 is available, which should greatly facilitate the identification of disease genes. One such candidate, CORNEO-DESMOSIN, is genetically linked to PSORS1 (mapping 160 kb distal to HLA-C) and is expressed in the skin with a proposed role in keratinocyte differentiation and desquamation. However, Nair et al.1xLocalisation of psoriasis-susceptibility locus PSORS1 to a 60-kb interval telomeric to HLA-C. Nair, R.P. et al. Am. J. Hum. Genet. 2000; 66: 1833–1844Abstract | Full Text | Full Text PDF | PubMed | Scopus (198)See all References1 now provide convincing evidence to exclude HLA-C and CORNEODESMOSIN as candidates for PSORS1.Using published genomic sequence and genome databases, the authors identified 62 polymorphic markers mapping to 6p21, which were then used to genotype families with at least one psoriatic individual. After analysis, the authors were able to identify an ancestral chromosome haplotype spanning 60 kb that is likely to harbour the same PSORS1 disease-associated mutation. This high-risk psoriasis haplotype was found in nearly two-thirds of all psoriasis-affected individuals in this study, suggesting that it is a major disease-associated mutation. Affected individuals that do not carry this high-risk haplotype might still carry the same mutation, but they might have inherited too little of the haplotype to be identified with this marker set. Alternatively, they might have inherited a distinct PSORS1 mutation or none at all.Nair et al. have defined a very small genetic interval that contains PSORS1, but which excludes HLA-C and CORNEODESMOSIN. This raises the questions of which gene does encode for PSORS1, what exactly is the high-risk mutation and why does it give an elevated risk of psoriasis? According to the gene map of this region, there are three genes that, although they are annotated as pseudogenes, need to be assessed. It might be that the entire 60 kb sequence of affected and multiple unaffected individuals needs to be compared before PSORS1 will be revealed. It is plausible that PSORS1 might not be a coding mutation but, instead, occurs within a promoter or long-range enhancer sequence that effects gene regulation and expression, for example, that of CORNEODESMOSIN. At present, PSORS1 remains elusive.

The complete DNA sequence of the O antigen gene region of Plesiomonas shigelloides serotype O17 which is identical to Shigella sonnei form I antigen.

We cloned and determined the sequence of a DNA region of approximately 15-kb containing the cluster of genes required for O17 antigen expression in the Escherichia coli K-12 strain from the chromosome of Plesiomonas shigelloides serotype O17:H2 strain. The sequencing analysis revealed that the minimum essential region of the P. shigelloides O17 antigen gene cluster had a size of approximately 11.5-kb and contained 9 contiguous open reading frames (ORFs), which were almost identical to the corresponding ORFs of Shigella sonnei form I antigen gene region, except for IS630 sequence, at the DNA as well as amino acid levels. The putative function of most of the ORFs could be determined on the basis of amino acid sequence similarities and characteristics. In addition, the G+C content of the P. shigelloides O17 antigen genes was lower than that of the chromosomal DNA of P. shigelloides and S. sonnei, suggesting that both P. shigelloides O17 and S. sonnei form I antigen genes had been derived from the same origin with a low G+C content.

Organization of Escherichia coli O157 O antigen gene cluster and identification of its specific genes.

The O157:H7 clone of Escherichia coli, which causes major, often prolonged outbreaks of gastroenteritis with hemolytic-uremic syndrome (HUS) such as those in Japan, Scotland, and the United States recently, is thought to be resident normally in cattle or other domestic animals. This clone is of major significance for public health and the food industry. We have developed a fast method for sequencing a given O antigen gene cluster and applied it to O157. The O157 O antigen gene cluster is 14 kb in length, comprising 12 genes and a remnant H-repeat unit. Based on sequence similarity, we have identified all the necessary O antigen genes, including five sugar biosynthetic pathway genes, four transferase genes, the O unit flippase gene, and the O antigen polymerase gene. By PCR testing against all 166 E. coli O serogroups and a range of gram-negative bacterial strains, including some that cross-react serologically with E. coli O157 antisera, we have found that certain O antigen genes are highly specific to O157 E. coli. This work provides the basis for a sensitive test for rapid detection of O157 E. coli. This is important both for decisions on patient care, since early treatment may reduce the risk of life-threatening complications, and for detection of sources of contamination. The method for fast sequencing of O antigen gene clusters plus an ability to predict which genes will be O antigen specific will enable PCR tests to be developed as needed for other clones of E. coli or, once flanking genes are identified, clones of any gram-negative bacterium.

Expression of the O antigen gene cluster is regulated by RfaH through the JUMPstart sequence

O antigen genes are clustered, with a JUMPstart sequence located upstream. JUMPstart is a 39-bp sequence, present upstream of many polysaccharide gene clusters and also upstream of haemolysin and F factor gene clusters. RfaH is known to regulate the expression of E. coli group II capsule, haemolysin, F factor and the outer core of lipopolysaccharide all of which have the JUMPstart sequence, and has been shown to function as a transcriptional antiterminator in some cases. Using lacZ fusions to genes in the O antigen gene cluster of Salmonella enterica serovar Typhimurium, we found that RfaH also regulates the expression of O antigen. We showed that RfaH enhances expression of the 18-kb O antigen gene cluster, with promoter-distal genes affected more dramatically. We also showed that the JUMPstart sequence was required for RfaH function.

Expression of the O antigen gene cluster is regulated by RfaH through the JUMPstart sequence.

O antigen genes are clustered, with a JUMPstart sequence located upstream. JUMPstart is a 39-bp sequence, present upstream of many polysaccharide gene clusters and also upstream of haemolysin and F factor gene clusters. RfaH is known to regulate the expression of E. coli group II capsule, haemolysin, F factor and the outer core of lipopolysaccharide all of which have the JUMPstart sequence, and has been shown to function as a transcriptional antiterminator in some cases. Using lacZ fusions to genes in the O antigen gene cluster of Salmonella enterica serovar Typhimurium, we found that RfaH also regulates the expression of O antigen. We showed that RfaH enhances expression of the 18-kb O antigen gene cluster, with promoter-distal genes affected more dramatically. We also showed that the JUMPstart sequence was required for RfaH function.

Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster.

The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied. Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS. Transcription of region 1 of the E. coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF. The promoter had -35 and -10 consensus sequences typical of an E. coli sigma 70 promoter, with no similarities to binding sites for other sigma factors. Two integration host factor (IHF) binding site consensus sequences were identified 110 bp upstream and 130 bp downstream of the transcription start site. In addition, two AT-rich regions separated by 16 bp identified upstream of the region 1 promoter were conserved upstream of the region 3 promoter. The kpsF gene was 98.8% identical with the kpsF gene identified in the E. coli K1 antigen gene cluster and confirms that the kpsF gene is conserved among group II capsule gene clusters. An intragenic Rho-dependent transcriptional terminator was discovered within the kpsF gene. No essential role for KpsF in the expression of the K5 antigen could be established. The temperature regulation of region 1 expression was at the level of transcription, with no transcription detectable in cells grown at 18 degrees C. Mutations in regulatory genes known to control temperature-dependent expression of a number of virulence genes had no effect on the temperature regulation of region 1 expression. Likewise, RfaH, which is known to regulate expression of E. coli group II capsules had no effect on the expression of region 1. Mutations in the himA and himD genes which encode the subunits of the IHF led to a fivefold reduction in the expression of KpsE at 37 degrees C, confirming a regulatory role for IHF in the expression of region 1 genes.

Sequence and analysis of the O antigen gene ( rfb) cluster of Escherichia coli 0111

The O antigens found in Salmonella enterica (Se) and Escherichia coli (Ec) show a great deal of diversity, and only three structures are known to be common to both genera. Two of them contain the 3,6-dideoxyhexose colitose, not found in other serogroups of the two species. The first of these is common to Ec O111 and Se 0:35 (sv Adelaide); the other is found in both Ec 055 and Se 0:50 (sv Greenside). The genes specific for the synthesis of O antigen are generally located in the rfb gene cluster at map position 45 min in Ec and 42 min in Se. The rfb (O antigen) gene cluster of an Ec O111 strain M92 had been cloned earlier and hybridisation analysis suggested that the rfb clusters of Ec M92 and a Se sv Adelaide strain had been acquired separately by the two species since their divergence. We have now sequenced part of the rfb cluster from Ec M92. We identify two genes of the GDP-colitose pathway, rfbM and rfbK, and show that several other ORFs have similarity to the rfb and cps (capsular polysaccharide) genes. Downstream of this block of genes is an ORF which encodes a protein with predicted transmembrane segments which is presumed to correspond to the rfbX gene. The % G + C values of the Ec M92 rfb sequence are extremely low, indicating that the rfb evolved in a low % G + C species of bacteria before transfer into Ec.

Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis.

Only Vibrio cholerae strains of serotype O1 are known to cause epidemics, while non-O1 strains are associated with sporadic cases of cholera. It was therefore unexpected that the recent cholera epidemic in Asia was caused by a non-O1 strain with the serotype O139. We provide evidence that O139 arose from a strain closely related to the causative agent of the present cholera pandemic, V. cholerae O1 El Tor, by acquisition of novel DNA which was inserted into, and replaced part of, the O antigen gene cluster of the recipient strain. Part of the novel DNA was sequenced and two open reading frames (otnA and otnB) were observed, the products of which showed homology to proteins involved in capsule and O antigen synthesis, respectively. This suggests that the otnAB DNA determines the distinct antigenic properties of the O139 cell surface. The otnAB DNA was not detected in O1 strains, but was present in two non-O1 V. cholerae strains with serotypes O69 and O141. In the O69 and O139 strains the otnAB genes were located proximate to the putative insertion sequence (IS) element rfbQRS, which is associated with O antigen synthesis genes in O1 strains, and may have played a role in the insertion of the otnAB DNA in the recipient chromosome. Our results suggest that the O139 strain arose by horizontal gene transfer between a non-O1 and an O1 strain. The acquired DNA has altered the antigenic properties of the recipient O1 strain, providing a selective advantage in a region where a large part of the population is immune to O1 strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster.

Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.

Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters.

Sequence and structural analysis of the rfb (O antigen) gene cluster from a group C1 Salmonella enterica strain.

The rfb (O antigen) gene cluster of a group C1 Salmonella enterica strain was sequenced; it comprised seven open reading frames which precisely replaced the 16 open reading frames of a group B strain. Two genes of the mannose biosynthetic pathway were present: rfbK (phosphomannomutase) had a G+C content of 0.61 and had only 40% identity to rfbK of group B but was very similar to cpsG of the capsular polysaccharide pathway with 96% identity, whereas rfbM [guanosine diphosphomannose (GDP-Man) pyrophosphorylase] had a G+C content of 0.39. Other genes had G+C contents ranging from 0.24 to 0.28. rfbM(C1) and rfbM(B) had 60% identity, which is much less than expected within a species, but nonetheless indicates a much more recent common ancestor than for rfbK. The other genes showed much lower or no similarity to rfb genes of other S. enterica strains. It appears that the gene cluster evolved outside of Salmonella in a species with low G+C content: the rfbM gene presumably derives from that period whereas the rfbK gene appears to have arisen after transfer of the cluster to S. enterica by duplication of the S. enterica cpsG gene, presumably replacing an rfbK gene of low G+C content.

Cloning and structure of group C1 O antigen (rfb gene cluster) from Salmonella enterica serovar montevideo.

The Salmonella enterica group C1 O antigen structure has a Man-Man-Man-Man-GlcNAc backbone with a glucose branch, which differs from the S. enterica group B O antigen structure which has a Man-Rha-Gal backbone with abequose as side-chain. We have cloned the group C1 rfb (O antigen) gene cluster from serovar montevideo strain M40, using a low-copy-number cosmid vector. The restriction map of the group C1 (M40) rfb gene cluster was compared with that of group B strain LT2 by Southern hybridization and restriction enzyme analysis. The results indicate that the flanking genes are very similar in the two strains, but there is no detectable similarity in the rfb regions. We localized the mannose pathway genes rfbM and rfbK and one of the genes, rfbK, shows considerably similarity to cpsG of strain LT2, suggesting that part of the mannose pathway in the group C1 rfb cluster is derived from a gene of the M antigen (cps) cluster. The M antigen, which forms a capsule, is comprised of four sugars, including fucose. The biosynthetic pathway of GDP-fucose has steps in common with the GDP-mannose pathway, and the cps cluster has isogenes of rfbK and rfbM, presumably as part of a fucose pathway. We discuss the structure and possible evolution of the group C1 rfb gene cluster.