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In Arthrobacter pyridinolis, a respiration-coupled transport system for L-rhamnose caused accumulation of free L-rhamnose, while a phosphoenolpyruvate: L-rhamnose phosphotransferase system caused accumulation of L-rhamnose I-phosphate (Levinson & Krulwich, 1974). The pathways for subsequent metabolism of L-rhamnose and L-rhamose I-phosphate have now been investigated. Arthrobacter pyridinolis contains an inducible L-rhamnose isomerase and L-rhamnulokinase, as well as a constitutive L-rhamnulose I-phosphate aldolase. Results with mutants which are unable to metabolize L-rhamnose suggest the presence of an L-rhamnose I-phosphate phosphatase, which forms free L-rhamnose by hydrolysis of L-rhamnose I-phosphate produced by the phosphotransferase system. Mutants which lack this enzyme exhibited severe inhibition of growth in the presence of L-rhamnose plus any of a variety of carbon sources. There is some evidence that this inhibition was due to accumulation of L-rhamnose I-phosphate at toxic concentrations within the bacteria. The metabolism of L-rhamnose transported by the phosphotransferase system therefore appears to occur by hydrolysis of L-rhamnose I-phosphate to free L-rhamnose by a phosphatase. Metabolism of the L-rhamnose thus produced, and of that accumulated by the respiration-coupled transport system, the proceeds by the sequence of reactions: L-rhamnose leads to L-rhamnulose leads to L=rhamnulose I-phosphate leads to dihydroxyacetone phosphate plus L-lactaldehyde.
Escherichia coli cannot grow on L-lyxose, a pentose analog of the 6-deoxyhexose L-rhamnose, which supports the growth of this and other enteric bacteria. L-Rhamnose is metabolized in E. coli by a system that consists of a rhamnose permease, rhamnose isomerase, rhamnulose kinase, and rhamnulose-1-phosphate aldolase, which yields the degradation products dihydroxyacetone phosphate and L-lactaldehyde. This aldehyde is oxidized to L-lactate by lactaldehyde dehydrogenase. All enzymes of the rhamnose system were found to be inducible not only by L-rhamnose but also by L-lyxose. L-Lyxose competed with L-rhamnose for the rhamnose transport system, and purified rhamnose isomerase catalyzed the conversion of L-lyxose into L-xylulose. However, rhamnulose kinase did not phosphorylate L-xylulose sufficiently to support the growth of wild-type E. coli on L-lyxose. Mutants able to grow on L-lyxose were analyzed and found to have a mutated rhamnulose kinase which phosphorylated L-xylulose as efficiently as the wild-type enzyme phosphorylated L-rhamnulose. Thus, the mutated kinase, mapped in the rha locus, enabled the growth of the mutant cells on L-lyxose. The glycolaldehyde generated in the cleavage of L-xylulose 1-phosphate by the rhamnulose-1-phosphate aldolase was oxidized by lactaldehyde dehydrogenase to glycolate, a compound normally utilized by E. coli.
L-rhamnose has been found to be useful to investigate the process of irreversible adsorption of PL-I phage to its host bacterium, Lactobacillus casei ATCC27092. L-rhamnose inhibited phage adsorption to cells without inactivating free phages. Adsorption inhibition was correlated with the concentrations of L-rhamnose. The inhibitory effect of L-rhamnose on phage adsorption was of a competitive nature against host cells. Among other saccharides tested, L-fucose, L-mannose and D-ribose showed a slight degree of adsorption-inhibiting activity. In early stages of phage adsorption in a tris-maleate buffer, where the binding of phages to cells was still reversible, addition of L-rhamnose resulted in the partial desorption of phages from the cells to which they had adsorbed. However, the number of infective phages desorbed by L-rhamnose treatment gradually fell off as incubation continued, showing that the phages became firmly bound to the cells. Therefore, it is possible to determine the number of phages irreversibly adsorbed to cells by using this desorption technique with L-rhamnose. The process of irreversible phage adsorption, that is, the formation of phage-cell complexes from which no more infective phages could be desorbed, was dependent on temperature and strongly inhibited at 0 degrees C.
A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp. cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate- and mutanolysin-treated cell walls. The cell wall carbohydrates of L. lactis subsp. cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine. Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, ml3, and 1 indicated that galactose was essential for binding all phages. In addition, rhamnose was required for binding phages kh and ml3. Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose. Rather, galactose may be an essential structural component located in the vicinity of the receptor. Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages. Inactivation required divalent cations and was irreversible. Inactivation of phages was stereospecific for rhamnose, as neither L-(+)- nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage. Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium. Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide.
A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-D-xylo-4-hexulose was isolated after a 1-h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-D-xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier. Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.
The coaggregation of the Gram-negative microorganism Capnocytophaga ochracea ATCC 33596 with several Gram-positive bacteria, including streptococci, actinomyces and rothia, is mediated by rhamnose-sensitive adhesin(s). In the present study, MAbs against the C. ochracea adhesin(s) were prepared. These antibodies inhibited all the rhamnose-sensitive interactions, indicating that they recognize epitopes at or near the rhamnose binding site of the adhesin. The monoclonals served as probes in immunoblot analysis and recognized a polypeptide of Mr 155 K present in the wild-type organism but absent in a coaggregation defective mutant. It is concluded that the rhamnose-sensitive interactions of C. ochracea are mediated by a 155 K Mr polypeptide present on its outer surface.
Metabolism and growth yields of Bacteroides ruminicola grown on d-xylose, l-arabinose, and l-rhamnose were studied. Growth yields were 62, 68, and 35.5 g (dry weight) per mol of carbohydrate fermented after correction for storage polysaccharide. Experiments with [1-(14)C]arabinose indicated that pentose was fermented by a pentose phosphate cycle plus glycolysis, with some indication of a minor phosphoketolase-type pathway. The product ratios from pentose were similar to those previously described for hexose. Rhamnose was fermented mainly to 1,2-propanediol, succinate, and acetate, although the latter was quantitatively less than expected. Estimates of adenosine 5'-triphosphate (ATP) molar growth yields could not be calculated with any certainty, as ATP generation by electron transport-linked phosphorylation cannot yet be assessed. If ATP were generated by substrate-level phosphorylation reactions alone, ATP molar growth yields for xylose, arabinose, and rhamnose would be 30, 28, and 35 g/mol. If calculations are based on an assumption that two ATP are generated by electron transport-linked phosphorylation per succinate, ATP molar growth yields become 15, 14, and 22 g/mol; if the assumption is also made that the pathway of lactaldehyde reduction is coupled to production of one ATP per 1,2-propanediol by electron transport-linked phosphorylation, the ATP molar growth yield for rhamnose fermentation becomes 14 g/mol. No preference can be expressed between these alternatives at present.
In the sera of patients with recurrent rheumocarditis, and especially in cases of primary rheumatism, the level of antibodies to group A streptococcal polysaccharide (A-PS) has been found, according to the results of the enzyme immunoassay, to be considerably higher than in the sera of healthy donors. The level of antibodies to rhamnose determinants (RD) of A-PS has been determined by the inhibition of the immunoenzyme reaction with A-PS under the influence of a variant of group A streptococcus and rhamnose disaccharides with the bonds alpha 1-2 and alpha 1-3. In patients with recurrent rheumocarditis the level of antibodies to A-PS has been shown to be considerably higher than in healthy donors having these antibodies. In acute primary rheumatism a high level of antibodies to A-PS has been detected only in a few cases, and at the same time the prevalence of antibodies to the specific RD of A-PS, bound with beta-N-acetylglucosamine, is observed. In the sera of patients with recurrent rheumocarditis and donors having a high content of antibodies to the rhamnose site of A-PS antibodies, seemingly active against at least two RD, have been detected. In acute primary rheumatism an insignificant amount of antibodies to the rhamnose site of A-PS may probably cause the autoimmune process accompanying rheumatism. This suggestion is substantiated by the previously established capacity of these antibodies for inducing the suppression of cytotoxic cell reactions to microbial antigens.
The kinetics of growth in vitro of strains Yersinia enterocolitica, serovars 0:3, 0:5, 0:8 and 0:9, on culture media containing rhamnose, raffinose and sodium succinate and at different temperatures of cultivation (4 degrees, 25 degrees and 37 degrees C) was characterized. It was established, that all the Yersinia enterocolitica serovars tested, cultivated at 25 degrees C, had the highest growth rate. A significant activation of growth at this temperature was observed when L-rhamnose and especially sodium succinate were used as substrates. The cultivation at 37 degrees C caused a sharp increase in the growth rate which, however, decreased at the end of the experiment (96th hour of cultivation), and certain stimulation of growth was observed on D-raffinose-containing medium. The growth of cells cultivated at 4 degrees C using L-rhamnose as substrate started after a lag-phase of 24 hours, which was better expressed for serovars 0:8 and 0:9. The results reported could be applied to experiments, using methods of isolation of Yersinia enterocolitica from different sources after cultivation at 4 degrees C on L-rhamnose-containing media.
The rhamnosyltransferase catalyzing the production of the bitter flavanone-glucosides, naringin and neohesperidin, was purified to homogeneity. The enzyme catalyzes the transfer of rhamnose from UDP-rhamnose to the C-2 hydroxyl group of glucose attached via C-7-O- of naringenin or hesperetin. To our knowledge this is the first complete purification of a rhamnosyl-transferase. The enzyme from young pummelo leaves was purified greater than 2,700-fold to a specific activity of over 600 pmol/min/mg of protein by sequential column chromatographies on Sephacryl S-200, reactive green 19-agarose, and Mono-Q. The enzyme was selectively eluted from the green dye column with only three other proteins by a pulse of the substrate hesperetin-7-O-glucoside followed by UDP. The rhamnosyltransferase is monomeric (approximately 52 kDa) by gel filtration and electrophoresis. The enzyme rhamnosylates only with UDP-rhamnose. Flavonoid-7-O-glucosides are usable acceptors but 5-O-glucosides or aglycones are not. It is inhibited by 10 microM UDP, its end product, but not by naringin or neohesperidin. Several flavonoid-aglycones at 100 microM inhibited the rhamnosyltransferase; UDP-sugars did not. The Km for UDP-rhamnose was similar with prunin (1.3 microM) and hesperetin-7-O-glucoside (1.1 microM) as substrate. The affinity for the natural acceptor prunin (Km = 2.4 microM) was much higher than for hesperetin-7-O-glucoside (Km = 41.5 microM). The isolation of the gene may enable its use in genetic engineering directed to modifying grapefruit bitterness.
dTDP-dihydrostreptose synthase from Streptomyces griseus was purfied about 50-fold by removal of protein with polyethyleneimine, (NH4)2SO4 fractionation and gel filtration on Ultrogel AcA44. The synthase preparation was free of dTDP-4-keto-L-rhamnose 3,5-epimerase (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, EC 5.1.3.13) activity. A new enzyme assay using Escherichia coli Y10 as source for the epimerase and dTDP-glucose 4,6-dehydratase (dTDP-glucose 4,6-hydro-lyase, EC 4.2.1.46) was developed. In the presence of excess epimerase the apparent Km for dTDP-4-keto-6-deoxy-D-glucose was determined to be 25 microM. The molecular weight of epimerase and synthase were determined by their elution volumes from a Sephadex G-100 column to be approx. 67,000 and 32,000, respectively. The pH optimum for the epimerase was between 7.5 and 8.5. The intermediate formation of dTDP-4-keto-L-rhamnose in the epimerase reaction could be shown by detection of 6-deoxy-[3H]talose after NaB3H4 reduction. Results which indicate the existence of dTDP-4-keto-6-rhamnose as a free intermediate in the epimerase reaction are reported.
The product of the rhaR gene, which regulates the level of mRNA produced from the four L-rhamnose-inducible promoters of the rhamnose operon, has been hypersynthesized and purified by a two-column procedure. The purified protein is a 33 kDa DNA-binding protein that binds to an inverted repeat structure located within the psr promoter, the promoter for the rhaS and rhaR genes. The equilibrium binding constants and kinetic constants have been determined under a variety of solution conditions. The protein binds with high affinity and its binding is sensitive to salt concentration and the presence of L-rhamnose. The nucleotides and phosphate residues contacted by RhaR were identified by chemical interference assays. All of the contacts are made to one face of the DNA and the symmetrical pattern matches the inverted repeat sequence proposed for the binding site. An unusual property of the binding site is that the two half-sites of the inverted repeat are separated from one another by 17 base-pairs of uncontacted DNA. Significant binding is retained if the 17 base-pairs are extended by insertions of integral turns of DNA, but not by half-integral turns. The complex of RhaR-DNA appears to be sharply bent, approximately 160 degrees.
A new acidic sugar, 3-O-[(R)-1-carboxyethyl]-L-rhamnose (1), has been identified as a constituent of the O-antigenic lipopolysaccharide of Sh. dysenteriae type 5. The structure of 1 has been established by physico-chemical methods and by synthesis. Alkylation of methyl 2,5-di-O-benzyl-alpha-L-rhamnofuranoside (6) with (S)- or (R)-2-chloropropionic acids, followed by removal of the protecting groups, afforded 3-O-[(R)-1-carboxyethyl]-L-rhamnose (9) and 3-O-[(S)-1-carboxyethyl]-L-rhamnose (10), respectively. The properties of 1 coincide with those of 9.
Complete assignments of the 13C-n.m.r. spectra of disaccharides having beta-glycosidic linkages are presented and discussed. The disaccharides of D-glucose, D-galactose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose are model compounds for 13C-n.m.r. studies of immunological polysaccharides. Changing the nature of the reducing glucopyranose rings (D-glucose to L-rhamnose) has no important influence on the chemical shifts of the carbons of the non-reducing glucopyranose ring (D-glucose). The converse is also true: the chemical shifts of the carbons of the reducing glucopyranose ring (L-rhamnose) are not noticeably affected by a change of the non-reducing unit (D-glucose to D-galactose or 2-acetamido-2-deoxy-D-glucose).
Thirteen atypical Yersinia enterocolitica isolates, all fermenting rhamnose, raffinose, and melibiose and utilizing sodium citrate within 24 to 48 h at 22 degrees C (Y.e.rh+), were examined biochemically-serologically, and by gas-liquid chromatography. These data, as well as cultural, biochemical, and antibiotic susceptibility data gathered from two previous studies involving (i) these same atypical Y.e.rh+ isolates, (ii) Y. enterocolitica serotypes O:1 through O:15 (rhamnose, raffinose, and citrate negative [Y.e.rh-]), (iii) Y. enterocolitica serotype O:16 (rhamnose positive but raffinose and citrate negative), and (iv) Yersinia pseudotuberculosis serogroups I through V were statistically compared. Both preand postabsorption agglutination studies demonstrated the serological distinctiveness of Y.e.rh+ from Y.e.rh- and Y. pseudotuberculosis. At the same time, three immunological groups among the 13 Y.e.rh+ strains were seen; 8 corresponded to Y. enterocolitica serotype O:17; 1 to Y. enterocolitica serotype O:16; and the remaining four were nontypable in antisera against known Y. enterocolitica antigen types. Each of the three Yersinia groups tested chromatographically produced acetic and lactic acids. Both Y.e.rh- and Y.e.rh+ formed propionic acid, but only Y.e.rh+ produced detectable amounts of succinic acid. Based on 49 variables, statistical analysis of the three Yersinia groups studied placed each of the Y.e.rh+ strains in a homogeneous group separate from both Y.e.rh- and Y. pseudotuberculosis. These data, coupled with deoxyribonucleic acid homology studies of Brenner and co-workers (D. J. Brenner, A. G. Steigerwalt, D. F. Falcao, R. E. Weaver, and G. R. Fanning, Int. J. Syst. Bacteriol. 26:180-194, 1976), support the distinctiveness of Y.e.rh+ from typical Y. enterocolitica and Y. pseudotuberculosis.
The main objective for a reinvestigation of rhamnose was to devise a mechanistic link between the trapped electron detected previously and the secondary radicals observed at 77 K and at room temperature. Single crystals of rhamnose were X-irradiated at temperatures between 15 and 300 K and examined using ESR, ENDOR, and field-swept ENDOR techniques. After low-temperature irradiation a C3 H-abstraction radical is formed following the visible light-induced decay of the trapped electron. This species was previously assigned erroneously to a C2 H-abstraction species. At temperatures above 120 K, this radical deprotonates at the C3 hydroxy group. Furthermore, a C2 H-abstraction radical is formed following the thermally induced decay of the trapped electron. The C2 and C3 H-abstraction radicals did not convert into each other. A third radical species formed at low temperatures is a C5 H-abstraction radical. It is unstable above 250 K and decays without any apparent successor. The C2 and C3 H-abstraction radicals are formed thermally and photochemically from the parent trapped electron. The conversions are mediated by hydrogen atoms formed intermediately or by elimination of hydride ions. The thermal decomposition pathway requires further studies, in particular with respect to the possible role of water. Recently, Box et al. analyzed the site of the trapped electron in rhamnose crystals. The present results support the results obtained by these authors (Radiat. Res. 121, 262 (1990)). In particular, trapped electron vs proton distances closely match the conversion mechanisms suggested.