Biosynthesis of the O Antigen from Citrobacter 139

The biosynthesis of the O antigen of Citrobacter 139 (Escherichia coli 3 Zurich 4,5,12:z(20)) was shown to proceed through a series of lipid-linked intermediates, similar to those involved in O-antigen synthesis in Salmonella. Galactose was the first sugar incorporated, followed by rhamnose and mannose. Abequose was incorporated from cytidine diphosphate (CDP)-abequose only when all three of the other nucleotide sugars (uridine diphosphate galactose, guanosine diphosphate mannose, and thymidine diphosphate rhamnose) were present. Rhamnosyl-galactosyl 1-phosphate and mannosyl-rhamnosyl-galactosyl 1-phosphate were identified as the products of mild alkaline hydrolysis of the lipid-linked intermediates.     

Cell wall lipopolysaccharide response to the ColIb plasmid mutants.

Mutants of ColIb plasmid affected the synthesis of O-side chains of lipopolysaccharides (LPS) in Salmonella. The plasmid srd 25 (defective in colicin synthesis) caused a significant decline of rhamnose and mannose content and lack of abequose in LPS of S. typhimurium. The number of repeating units in O-side chains was decreased after the indroduction of srd 25. Cultures of S. typhimurium and S. enteritidis harboring drd2 (derepressed in colicin production) polymerised dideoxyhexose-defective O-side chains i.e. deprived of abequose and tyvelose, respectively. In dideoxyhexoseless S. meleagridis the content of rhamnose and mannose were reduced. The information for the alterations of Salmonella LPS was contained in the plasmid genome. In the wild-type plasmids the genes controlling the O-antigen changes were not expressed.     

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.     

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.     

Enterobacterial common antigen in rfb deletion mutants of Salmonella typhimurium.

The his-rfb deletion series of Salmonella typhimurium mutants characterized previously by Nikaido et al. was examined for the presence of the enterobacterial common antigen (ECA). All deletions not extending further to the left than the genes for cytidine phosphoabequose synthesis were ECA positive, whereas longer deletions (extending to the genes for thymidine diphosphorhamnose synthesis or further) were ECA negative. When these long-his-rfb deletion strains were studied further, it became clear that they (four out of four studied) had accumulated a second mutation, called rff, close to ilv, which prevented the synthesis of ECA. When rff- was replaced by rff+, the recombinants, now having the his-rfb deletion only, produced traces of ECA, showed reduced viability, increased sensitivity to sodium dodecyl sulfate (SDS) and to a lesser extent, to other anionic detergents, and accumulated secondary "suppressor" mutations upon storage. Such suppressor-containing mutants could be isolated by selecting for resistance to 1% SDS. Thirty of 46 SDS-resistant mutants studied had a second mutation, which alone prevented the synthesis of ECA, close to ilv. This ilv-linked mutation was similar to the rff mutation of the strains studied originally. The new rff mutation was similar to previously described rfe mutations in its close linkage to ilv and association with an ECA-negative phenotype. It differed from rfe, however, by not affecting the synthesis of the O antigens (O-6,7) of group C1. In Salmonella group C1, all ECA genes identified thus far are linked to ilv (rfe and/or rff) and none is linked to rfb.     

Phagocytosis of bacteria by macrophages: changing the carbohydrate of lipopolysaccharide alters interaction with complement and macrophages

Salmonella transductants and recombinants differing the O-antigenic side chain of their lipopolysaccharide are taken up at different rates by the murine macrophage-like cell line J774. Bacteria containing abequose, mannose, rhamnose, and galactose in O-antigenic side chain were taken up at the slowest rate; the one containing tyvelose instead of abequose was taken up at an intermediate rate; and the one containing mannose, N-acetylglucosamine, and glucose, instead of the above sequence, was taken up at the highest rate. These rates correlate well with the known virulence of these strains; the most virulent is the one taken up slowest, the one taken up at an intermediate rate is less virulent, and the one taken up fastest is the least virulent. The differences in ingestion rates reflect differences in affinity of the bacteria for the macrophages and not in the rate of ingestion once interaction has occurred, suggesting a receptor-mediated process. The majority of uptake is probably dependent on complement, as shown by the requirement for a serum component(s) destroyed by heating at 56 degrees C or by incubation with zymosan. Specific antibody is not required. We therefore postulate that relative virulence in vivo may reflect the relative ability of the polysaccharide of bacterial lipopolysaccharide to activate complement, thus determining the susceptibility of the bacteria to ingestion via the complement receptor of phagocytic cells.     

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.     

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.     

Identification and sequence of rfbS and rfbE, which determine antigenic specificity of group A and group D salmonellae.

Salmonella group A, group B, and group D strains have paratose, abequose, and tyvelose, respectively, as the immunodominant sugar in their O antigens, which are otherwise identical; only the final steps differ in the biosynthetic pathways of these sugars. The gene rfbJ from a group B strain, encoding abequose synthase, the final and only unique step in the biosynthesis of CDP-abequose, has been cloned and sequenced (P. Wyk and P. Reeves, J. Bacteriol. 171:5687-5693, 1989). In this study, we locate and sequence rfbS and rfbE from serovars typhi and paratyphi, representative of groups A and D. Gene rfbS is present in both groups and encodes paratose synthase, which carries out a step parallel to that of abequose synthase, but the product is CDP-paratose. The DNA and inferred amino acid sequences are compared with those of rfbJ. We conclude that the genes are homologous, but the divergence is extremely ancient. Gene rfbE encodes CDP-tyvelose epimerase, which converts CDP-paratose to CDP-tyvelose in group D strains; the gene is active in group D strains, and we find it to be present in a mutant form in group A strains. These two genes encode the steps unique to groups A and D and, like rfbJ of group B, are of low G+C content, suggesting transfer from outside of salmonellae. The evolutionary origin of these genes is discussed.     

Lipopolysaccharide composition of three strains of Haemophilus influenzae

The lipopolysaccharides of three strains of Haemophilus influenzae with varying beta-lactam susceptibility were examined. All three strains contained galactose, glucose, galactosamine, glucosamine, heptose, phosphate, and a trace of mannose. None contained fucose, rhamnose, or mannosamine. Levels of 2-keto-3-deoxy-octulosonic acid were consistently detected in all three strains at levels similar to that of Salmonella typhimurium LT2, but only following hydrolysis with 4 N hydrochloric acid.     

Chemically synthesized galactosyl ficaprenyl diphosphate as an intermediate in the biosynthesis of the Salmonella O-antigenic polysaccharide

The membrane fraction from a mutant of Salmonella anatum deficient in UDPgalactose-4-epimerase, utilized synthetic ficaprenyl alpha-D-galactosyl diphosphate as a substrate in the biosynthesis of the O-polysaccharide portion of lipopolysaccharide which has a mannosylrhamnosylgalactose repeating sequence. The galactosyl lipid was prepared by chemical synthesis from D-galactose and ficaprenol extracted from Ficus elastica. Membrane preparations catalyzed the transfer of rhamnose from TDP-rhamnose onto membrane-bound ficaprenyl galactosyl diphosphate forming rhamnosylgalactosyl ficaprenyl diphosphate; the reaction was dependent on the prior insertion of the synthetic glycosyl-lipid into the membrane, and was proportional to incubation time up to 4 min at 29 degrees C. When both TDP-rhamnose and GDP-mannose were added, the product formed was O-polysaccharide. These results indicate that the chemically synthesized ficaprenyl galactosyl diphosphate can be an active substrate for the in vitro synthesis of the Salmonella O-polysaccharide.     

尿苷二磷酸糖固定化酶合成法研究

利用生物酶进行体外催化反应合成不同种类的尿苷二磷酸糖（uridine diphosphate sugar,UDP-糖）,生物酶的重复利用率较低。为提高尿苷二磷酸糖的合成效率及增加产物种类,以镍螯合聚丙烯酸酯树脂为载体,对带有HIS标签的N-乙酰己糖胺激酶（N-acetylhexosamine kinase,NahK）和尿苷转移酶（uridine transferase,GlmU）进行固定化。以固定化NahK和固定化GlmU为催化酶,不同单糖作为底物,研究尿苷二磷酸糖的一锅法合成情况。利用Q柱对产物进行纯化,通过高效液相色谱法、质谱法、核磁共振氢谱法对反应产物进行检测。确定了镍螯合聚丙烯酸酯树脂对游离NahK和GlmU的实际载量分别为10和20 mg·g^-1。固定化酶量的最优配比为5.5 g固定化NahK和2.5 g固定化GlmU。固定化酶的最适pH和温度分别为8.0和35℃,且能在重复反应中稳定反应5个批次。葡萄糖、N-乙酰氨基葡萄糖和甘露糖可以参与一锅法反应,生成UDP-糖的相对分子质量分别为566、607、566,而葡萄糖醛酸、半乳糖和果糖在该体系下不能合成相应的UDP-糖。基于固定化酶技术,一锅法可合成UDP-葡萄糖、UDP-N-乙酰氨基葡萄糖、UDP-甘露糖。     

Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides.

Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They possessed kinases for deoxycytidine, (d)CMP, thymidine (deoxyuridine), dTMP, and nucleoside diphosphates; dCTPase and dUTPase; dCMP deaminase; thymidine (deoxyuridine) phosphorylase; and dUMP (dTMP) phosphatase. The existence of these enzymic activities together with ribonucleoside diphosphate reductase explains the capacity of cytidine to provide M. mycoides with deoxyribose for the synthesis of thymidine nucleotides from thymine.     

Repression of cytidine triphosphate synthetase in Salmonella typhimurium by pyrimidines during uridine nucleotide depletion

Regulation of the synthesis of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2) was investigated in Salmonella typhimurium. CTP synthetase appeared to be repressed only when intracellular concentrations of uridine nucleotides were significantly lowered. Under such nucleotide pool conditions, a cytidine compound and, to a lesser degree, a thymidine compound appeared as putative repressing metabolites of enzyme synthesis.     

Rhamnose synthase activity is required for pathogenicity of the vascular wilt fungus Verticillium dahliae

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so‐called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide‐rhamnose synthase/epimerase‐reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy‐thymidine diphosphate (dTDP)‐rhamnose, a precursor of L‐rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal–host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)‐rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.     

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     

Urease-negative strains of Clostridium sordellii.

Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C. sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose. The test for cell wall sugar components of urease-negative and -positive strains of C. sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.     

Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1).

The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.     

Chemotaxonomic study of Vibrio cholerae bio-serogroup Hakata on the basis of the sugar composition of the polysaccharide portion of its lipopolysaccharides

A chemotaxonomic study was carried out on 31 strains of non-O1 Vibrio cholerae bio-serogroup Hakata, isolated in Japan, which possesses the Inaba antigen C of O1 V. cholerae. On the basis of the compositional sugar pattern of the polysaccharide portion of their lipopolysaccharides, the 23 strains isolated from the environment were separated into two groups, one (20 strains) containing mannose, glucose, fructose, L-glycero-D-mannoheptose, glucosamine, perosamine, quinovosamine, and an unidentified amino sugar AS, and the other (3 strains) containing two additional sugars, galactose and a trace amount of galactosamine. All of the eight strains isolated from imported seafoods belonged to the former group.     

L-Rhamnose utilisation in Salmonella typhimurium

A khy , M.T., B rown , C.M. & O ld , D.C. 1984. L-Rhamnose utilisation in Salmonella typhimurium. Journal of Applied Bacteriology 56 , 269–274.
L-Rhamnose is degraded by strains of Salmonella typhimurium by isomerisation to L-rhamnulose, phosphorylation to L-rhamnulose-1-phosphate and cleavage to lac-taldehyde and dihydroxyacetone phosphate. The enzymes involved are, respectively, rhamnose isomerase (Rhal), rhamnulokinase (RhuK) and an aldolase (Ald). Strains able to grow rapidly on L-rhamnose contained a high-affinity uptake system for ³H-L-rhamnose that was induced by L-rhamnose and repressed by D-glucose. The synthesis of Rhal and RhuK was also induced by L-rhamnose but was not repressed by D-glucose. The synthesis of Ald was constitutive. Data are presented on some strains which grow very slowly on L-rhamnose and on others which do not utilise it.