Identification and sequence of the gene for abequose synthase, which confers antigenic specificity on group B salmonellae: homology with galactose epimerase.

The O antigen of Salmonella group B strains contains the sugar abequose, whereas those from group A and D strains contain paratose or tyvelose in its place. This is the essential difference between these Salmonella groups. Only the final step in the biosynthesis of abequose differs from that of paratose, and the abequose confers on group B strains their specific O4 antigen. The gene, rfbJ, encoding the enzyme abequose synthase for this last specific step has been cloned, identified, and sequenced and has been shown to function in group A and D strains to make them O4+. This one gene thus differentiates group B from group A or group D salmonellae. The enzyme abequose synthase appears to be related to galactose epimerase, and the significance of this is discussed. The rfbJ gene and adjacent DNA is of much lower G+C content than is usual for salmonellae, indicating that the region did not originate in a salmonella but was transferred from outside.     

Relationships among the rfb regions of Salmonella serovars A, B, and D.

The O antigens of Salmonella serogroups A, B, and D differ structurally in their side chain sugar residues. The genes encoding O-antigen biosynthesis are clustered in the rfb operon. The gene rfbJ in strain LT2 (serovar typhimurium, group B) and the genes rfbS and rfbE in strain Ty2 (serovar typhi, group D) account for the known differences in the rfb gene clusters used for determination of group specificity. In this paper, we report the nucleotide sequence of 2.9 kb of DNA from the rfb gene cluster of strain Ty2 and the finding of two open reading frames which have limited similarity with the corresponding open reading frames of strain LT2. These two genes complete the sequence of the rfb region of group D strain Ty2 if we use strain LT2 sequence where restriction site data show it to be extremely similar to the strain Ty2 sequence. The restriction map of the rfb gene cluster in group A strain IMVS1316 (serovar paratyphi) is identical to that of the cluster in strain Ty2 except for a frameshift mutation in rfbE and a triplicated region. The rfb gene clusters of these three strains are compared, and the evolutionary origin of these genes is discussed.     

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.     

Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B

The rfb (O antigen) gene cluster of group C2 Salmonella differs from that of group B in a central region of 12.4 kb: we report the sequencing of this region of strain M67 (group C2) and a subsequent comparison with the central region of strain LT2 (group B). We find a block of seven open reading frames unique to group C2 which encode the O antigen polymerase (rfc) and the transferases responsible for assembly of the group C2 O antigen. The remaining rfb genes are common to strains M67 and LT2, but rfbJ (CDP-abequose synthase) and rfbM and rfbK (GDP-mannose synthesis), which are immediately adjacent to the central region, are highly divergent. All these genes have a low G+C content and appear to have been recent additions to Salmonella enterica. We discuss the evolutionary significance of the arrangement and divergence of the genes in the polymorphism of the rfb cluster.     

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.     

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.     

Genetic determination of enzymes synthesizing O-specific sugars of Salmonella lipopolysaccharides

Nikaido, Hiroshi (Massachusetts General Hospital, Boston, Mass.), Kishiko Nikaido, and P. Helena M?kel?. Genetic determination of enzymes synthesizing O-specific sugars of Salmonella lipopolysaccharides. J. Bacteriol. 91:1126-1135. 1966.-Levels of enzymes involved in the biosynthesis of various nucleotide sugars were examined in parental strains and recombinants obtained in crosses between Salmonella of groups B, C(2), and C(1) with the O antigen specificities 4, 5, 12; 6, 8; and 6,7, respectively. The results showed that smooth strains of groups B and C(2) possessed the enzymes for the synthesis of guanosine diphosphate mannose, cytidine diphosphate abequose, and thymidine diphosphate rhamnose; these sugars are constituents of their lipopolysaccharides. Group C(1) lipopolysaccharide is devoid of both abequose and rhamnose, and the corresponding enzymes for cytidine diphosphate abequose synthesis, as well as the enzyme(s) catalyzing the last step(s) of thymidine diphosphate rhamnose synthesis, were undetectable in S. montevideo of this group. Two other enzymes also involved in the biosynthesis of thymidine diphosphate rhamnose were present at a low level of activity; their function in this strain is not known. The analysis of enzyme levels in recombinants indicated that genes determining at least eight of the enzymes involved in the biosynthesis of nucleotide-bound mannose, rhamnose, and abequose were located in the O locus known to determine the specificity of the O antigen. In three rough recombinant strains, enzyme levels indicated that crossing-over had presumably occurred within the O locus. The results also suggested a high degree of nonhomology in this region of the chromosome between groups B and C(1).     

Enhanced persistence in the colonic microbiota of Escherichia coli strains belonging to phylogenetic group B2: role of virulence factors and adherence to colonic cells

Escherichia coli segregates into four phylogenetic groups, A, B1, B2 and D. B2 and D strains usually possess virulence factors, cause most extra-intestinal infections and have superior capacity to persist in the infantile colonic microbiota. Here, we investigated 24 resident and 37 transient E. coli strains from the colonic microbiota of 13 Swedish schoolgirls sampled in the 1970s with respect to phylogenetic group identity, carriage of virulence factor genes, O and K antigens and mannose-sensitive and -resistant adherence to the colonic cell line HT-29. Resident strains more often belonged to phylogenetic group B2 than transient strains (38% vs 5% p=0.004). In contrast, transient strains more often than resident strains belonged to group A (57% vs 29%, p=0.04) or B1 (24% vs 13%, p=0.33). Most B2 strains belonged to uropathogenic O serogroups, carried genes for P fimbriae, K5 capsule and hemolysin and adhered in higher numbers to HT-29 cells via mannose-resistant mechanisms than strains from the other groups. Further, among strains carrying genes for P or S fimbriae, those belonging to group B2 adhered in highest numbers. In logistic regression, genes for P fimbriae and aerobactin predicted persistence in the colonic microbiota (p=0.050 and 0.056, respectively), while B2 origin did not reach significance as an independent variable (p=0.16). Our results indicate that virulence factors carried by group B2 strains contribute to their strong colonizing capacity. These factors may actually be regarded as fitness factors in the human gut.     

Studies of the biosynthesis of 3,6-dideoxyhexoses: molecular cloning and characterization of the asc (ascarylose) region from Yersinia pseudotuberculosis serogroup VA.

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to occur naturally, four have been found in various strains of Salmonella enterica (abequose, tyvelose, paratose, and colitose) and all five, including ascarylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region harboring the abequose biosynthetic genes from Y. pseudotuberculosis serogroup HA, the detailed genetic principles underlying a 3,6-dideoxyhexose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dideoxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (O antigen) cluster of Salmonella spp., we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a detailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In addition, the functional cloning of this region has allowed the expression of Ep (alpha-D-glucose cytidylyltransferase), Eod (CDP-D-glucose 4,6-dehydratase), E1 (CDP-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase), E3 (CDP-6-deoxy-delta 3,4-glucoseen reductase), Eep (CDP-3,6-dideoxy-D-glycero-D- glycero-4-hexulose-5-epimerase), and Ered (CDP-3,6-dideoxy-L-glycero-D-glycero-4-hexulose-4-reductase), facilitating future mechanistic studies of this intriguing biosynthetic pathway.     

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.     

Rapid and simple determination of the Escherichia coli phylogenetic group

Phylogenetic analysis has shown that Escherichia coli is composed of four main phylogenetic groups (A, B1, B2, and D) and that virulent extra-intestinal strains mainly belong to groups B2 and D. Actually, phylogenetic groups can be determined by multilocus enzyme electrophoresis or ribotyping, both of which are complex, time-consuming techniques. We describe a simple and rapid phylogenetic grouping technique based on triplex PCR. The method, which uses a combination of two genes (chuA and yjaA) and an anonymous DNA fragment, was tested with 230 strains and showed excellent correlation with reference methods.     

Mutants of Group D1Salmonella Carrying the Somatic Antigen of Group A Organisms: Isolation and Serological Characterization

O antigen mutants were obtained from Salmonella durban, a group D(1) organism, by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Serological studies demonstrated that the mutants lost the O-9 antigen factor of the parent organism but acquired the O-2 factor specific to group A Salmonella. Lipopolysaccharides of the mutant strains contained paratose which determines the specificity of O-2 factor. Tyvelose, present in the wild-type lipopolysaccharide, was not found in the mutants. H antigens and other biological characteristics of the mutant strains were the same as those of the wild-type organism. The present finding implies that group A Salmonella species might be derived from group D(1) organisms.     

Comparative genetic study of group B streptococcal strains of human and bovine origin

The presence and restriction fragment length polymorphism (RFLP) of DNA fragments hybridizing with virulence and "house keeping" gene probes were analyzed for 87 group B streptococcal (GBS) strains of human and bovine origin. Most characteristics obtained for bovine strains were similar when compared with those for human strains. The most significant degree of RFLP was discovered for the sizes of HindIII fragments containing bca gene. Human GBS strains with bac gene, encoding beta antigen with IgA binding capacity, were characterized by almost identical complex hybridization patterns with multiple gene probes. At the same time bac gene was not found among bovine GBS strains. Gene scpB that encodes C5a peptidase in all human GBS strains was detected only in 9 of 39 strains of bovine origin. These two characteristics effectively distinguished bovine GBS strains from GBS strains of human origin.     

Synthesis of stereospecifically labeled 3,6-dideoxyhexoses

Preparations of ascarylose (3,6-dideoxy-L-arabino-hexose), abequose (3,6-dideoxy-D-xylo-hexose), and paratose (3,6-dideoxy-D-ribo-hexose) with stereospecific deuterium labeling at C-3 are discussed. The methods used to synthesize these sugars, such as the hydrogenation of olefins, the displacement of halides, the reduction of epoxides, and the substitution of tosyl esters, illustrate a variety of strategies leading to stereospecific deuterium incorporation. Many of the techniques described here should be of general utility for the synthesis of other deuterium-labeled sugars.     

Mutants of Group D1Salmonella Carrying the Somatic Antigen of Group A Organisms: Evidence for the Lack of Cytidine Diphosphate Paratose-2-Epimerase Activity

The mutant strains of Salmonella durban that possessed O antigen 2, 12 of group A Salmonella were defective in the cytidine diphosphate paratose-2-epimerase activity. The enzyme preparation of the mutant strains catalyzed the conversion of cytidine diphosphate glucose into cytidine diphosphate paratose but not into cytidine diphosphate tyvelose. The defect in the epimerase activity was also confirmed by the use of purified cytidine diphosphate paratose as a substrate. The specificity of dideoxyhexosyl transferase catalyzing the formation of the group-specific determinant is discussed.     

Silencing of a second dimethylallyltryptophan synthase of <Emphasis Type="Italic">Penicillium roqueforti</Emphasis> reveals a novel clavine alkaloid gene cluster

Penicillium roqueforti produces several prenylated indole alkaloids, including roquefortine C and clavine alkaloids. The first step in the biosynthesis of roquefortine C is the prenylation of tryptophan-derived dipeptides by a dimethylallyltryptophan synthase, specific for roquefortine biosynthesis (roquefortine prenyltransferase). A second dimethylallyltryptophan synthase, DmaW2, different from the roquefortine prenyltransferase, has been studied in this article. Silencing the gene encoding this second dimethylallyltryptophan synthase, dmaW2, proved that inactivation of this gene does not prevent the production of roquefortine C, but suppresses the formation of other indole alkaloids. Mass spectrometry studies have identified these compounds as isofumigaclavine A, the pathway final product and prenylated intermediates. The silencing does not affect the production of mycophenolic acid and andrastin A. A bioinformatic study of the genome of P. roqueforti revealed that DmaW2 (renamed IfgA) is a prenyltransferase involved in isofumigaclavine A biosynthesis encoded by a gene located in a six genes cluster (cluster A). A second three genes cluster (cluster B) encodes the so-called yellow enzyme and enzymes for the late steps for the conversion of festuclavine to isofumigaclavine A. The yellow enzyme contains a tyrosine-181 at its active center, as occurs in Neosartorya fumigata, but in contrast to the Clavicipitaceae fungi. A complete isofumigaclavines A and B biosynthetic pathway is proposed based on the finding of these studies on the biosynthesis of clavine alkaloids.     

Three subfamilies of pheromone and receptor genes generate multiple B mating specificities in the mushroom Coprinus cinereus

The B mating type locus of the basidiomycete Coprinus cinereus encodes a large family of lipopeptide pheromones and their seven transmembrane domain receptors. Here we show that the B42 locus, like the previously described B6 locus, derives its unique specificity from nine multiallelic genes that are organized into three subgroups each comprising a receptor and two pheromone genes. We show that the three genes within each group are kept together as a functional unit by being embedded in an allele-specific DNA sequence. Using a combination of sequence analysis, Southern blotting, and DNA-mediated transformation with cloned genes, we demonstrate that different B loci may share alleles of one or two groups of genes. This is consistent with the prediction that the three subgroups of genes are functionally redundant and that it is the different combinations of their alleles that generate the multiple B mating specificities found in nature. The B42 locus was found to contain an additional gene, mfs1, that encodes a putative multidrug transporter belonging to the major facilitator family. In strains with other B mating specificities, this gene, whose functional significance was not established, lies in a region of shared homology flanking the B locus.     

Construction and characterization of isogenic O-antigen variants of Salmonella typhi

A 7.5 kb Kpnl-generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild-type Salmonella typhi Vi-positive strain ISP 1820 (i.e. serotype 09,12; Vi⁺ H-d), S. typhi Vi-negative strain H400 (i.e. serotype 09,12; Vi⁻; H-d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906-O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906-O4 demonstrated that these 8trains express the 04 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi⁺; H-d and the serotype of H404 is O4,12; Vi⁻; H-d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906-O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate-tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906-O4. The resistance of each strain to the bactericidal effects of guinea-pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC. The results suggest that the development of the O-antigen serotype diversity of Salmonella is probably the result of both sequence divergence and recombination     

ABO blood group system

The ABO blood group system in humans has three different carbohydrate antigens named A, B, and O. The A antigen sequence is terminal trisaccharide N-acetylgalactosamine (GalNAc)α1-3[Fucα1-2]Galβ-, B is terminal trisaccharide Galα1-3[Fucα1-2]Galβ-, and O is terminal disaccharide Fucα1-2Galβ-. The single ABO gene locus has three alleles types A, B and O. The A and B genes code A and B glycosyltransferases respectively and O encodes an inactive enzyme. A large allelic diversity has been found for A and B transferases resulting in the genetic subgrouping of each ABO blood type. Genes for both transferases have been cloned and the 3D structure of enzymes with and without substrate has been revealed by NMR and X ray crystallography. The ABO blood group system plays a vital role in transfusion, organ and tissue transplantation, as well as in cellular or molecular therapies.     

A modified overlap extension PCR method to create chimeric genes in the absence of restriction enzymes

A modified overlap extension technique for the creation of chimeric genes is described: the method consists in three PCR steps. The first step is a conventional PCR reaction, in which oligonucleotide primers are partially complementary at their 5' ends to the adjacent fragments that are fused to create the chimer. The second PCR step consists in the fusion of the PCR fragments generated in the first step using the complementary extremities of the primers. The third step corresponds to the PCR amplification of the fusion product. The final PCR product is a chimeric gene built up with the different amplified PCR fragments. The technique is illustrated by the construction of a chimeric 5- hydroxytryptamine (5-HT, serotonin)1B/D receptor by combining one part of the human 5-HT1B (h5-HT1B) and two parts of the h5-HT1D receptor gene. The chimeric gene expressed in Cos-7 cells yielded similar binding properties as the wild type h5-HT1D receptor. © Rapid Science Ltd. 1998