On the regulation of D-ribose metabolism in E. coli B-r. I. Isolation and characterization of D-ribokinaseless and D-ribose permeaseless mutants

Summary A presumed single site mutation designated leu-500, affects both the basal expression and response to the leucine repression signal by the leucine operon of Salmonella typhimurium. The distantly located supX mutation suppresses the leucine auxotrophy imposed by the leu-500 mutation by raising the level of basal expression while maintaining the abnormal regulation. An additional type of suppressor mutation which is closely linked to the leu-500 locus restores essentially normal regulation but maintains the low repressed expression characteristic of the leu-500 strain. The leucine sufficiency of the leu-500 strain with the linked suppressor and the supX leu-500 strains is temperature conditional in that both types require leucine for growth at 42° but not at 37°. These results, which indicate that a single site mutation can simultaneously affect promoter-like and operator-like function, are discussed in terms of DNA superstructure.This investigation was supported by U.S. Public Health Service Research Grant GM-12551 from the National Institute of General Medical Sciences, and is a portion of the work submitted by Lloyd H. Graf in partial fulfillment of the requirements for the hP. D. degree from Duke University. Lloyd H. Graf was the recipient of a predoctoral fellowship award from the National Institute of General Medical Sciences. R. O. Burns is the recipient of a Research Career Development Award from the National Institutes of General Medical Sciences.     

D-核糖高产菌株的选育

以肌苷产生菌B3为出发菌株,通过硫酸二乙酯,紫外线及原生质体紫外线等复合诱变处理,获得一株核糖高产积累突变株B1916,对突变株B1916遗传进行了研究,结果发现该突变株对碳水化合物的利用能力发生了变化,如对葡萄糖利用能力下降,不能利用阿拉伯糖和葡萄糖酸,丧失了解子形成能力;在培养过程中细胞形态呈链状,数字状等。     

发酵法生产D-核糖

Ｄ－核糖是一种重要的生理物质,可用于合成维生素Ｂ２,风味提高剂以及多种核酸药物等,具有广泛的应用前景,由于化学合成法存在污染公害,故近年来各国相继研究微生物发酵法生产Ｄ－核糖,并致力于工业化生产。本文对国内外Ｄ－核糖的研究进展作了综述,并对Ｄ－核糖的提取方法进行了探讨。     

Evolution of the D-ribose operon on Escherichia coli B/r.

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.     

Chiral histidine selection by D-ribose RNA

The invariant choice of L-amino acids and D-ribose RNA for biological translation requires explanation. Here we study this chiral choice using mixed, equimolar D-ribose RNAs having 15, 18, 21, 27, 35, and 45 contiguous randomized nucleotides. These are used for simultaneous affinity selection of the smallest bound and eluted RNAs using equal amounts of L- and D-His immobilized on an achiral glass support, with racemic histidine elution. The experiment as a whole therefore determines whether RNA containing D-ribose binds L-histidine or D-histidine more easily (that is, by using a site that is more abundant/requires fewer nucleotides). The most prevalent/smallest RNA sites are reproducibly and repeatedly selected and there is a four- to sixfold greater abundance of L-histidine sites. RNA's chiral D-ribose therefore yields a more frequent fit to L-histidine. Accordingly, a D-ribose RNA site for L-His is smaller by the equivalent of just over one conserved nucleotide. The most prevalent L-His site also performs better than the most frequent D-His site-but rarer D-ribose RNAs can bind D-His with excellent affinity and discrimination. The prevalent L-His site is one we have selected before under very different conditions. Thus, selection is again reproducible, as is the recurrence of cognate coding triplets in these most probable L-His sites. If our selected RNA population were equilibrated with racemic His, we calculate that L-His would participate in seven of eight His:RNA complexes, or more. Thus, if D-ribose RNA were first chosen biologically, translational L-His usage could have followed.     

添加物对D-核糖生物合成的影响

采用枯草杆菌Ｄ２０１转酮醇酶缺陷型突变株,研究菌种生长与Ｄ－核糖合成优化工艺。在培养剂中添加山梨醇、ＫＨ２ＰＯ４和芳香族氨基酸,发现对菌的生长有刺激作用,特别是山梨醇,它可缩短延迟期,促进菌的生长。此外,用葡萄糖酸钙部分代替葡萄糖能增加Ｄ－核糖的产量,用葡萄糖和葡萄糖酸的混合碳源合碳源发酵,Ｄ－核糖产量可达到５２ｇ／ｌ。     

The entry of D-ribose into some yeasts of the genus Pichia.

The utilization of D-ribose by yeasts of the genus Pichia was examined with respect to aerobic growth, respiration and entry of ribose into the cells. Pichia etchellsii (CBS2011) could respire D-ribose, but not use it for aerobic growth. Pichia fermentans (CBS187) neither respired nor grew on D-ribose, though it entered the cells of this yeast either by simple diffusion, or possibly, by the D-glucose carrier, this having a very low affinity for D-ribose. Pichia pinus (CBS5097) respired and grew on D-ribose; kinetic evidence is given for this yeast having two ribose carriers, one inducible and the other constitutive.     

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.     

几种添加物对D—核糖产量的影响

研究了几种物质对D－核糖产量的影响,研究表明,当发酵培养基中玉米浆的含量为25g/L,D－核糖产量达到最高,为65g/L。发酵培养基中添加适量的粉及山梨醇亦有利于D－核糖的积累,当粉与山梨醇的添加量分别为10g/L及40g/L时,D－核糖产率分别增加7.8%、4.7%,而在发酵培养基中加入丙二酸可抑制D－核的分泌,在40ml发酵培养基中添加1ml丙二酸后,D－核糖的产率下降73.8％。甲醇也可抑制D－核糖的积累,当发酵培养基中的甲醇添加量为16g/L时,D－核糖产率下降66.2%。     

On the regulation of D-ribose metabolism in E. coli B-r. II. Chromosomal location and fine structure analysis of the D-ribose permease and D-ribokinase structural genes by P 1 transduction

Summary The genetic mapping and fine structure analysis of the d-ribose gene in Escherichia coli B/r has been studied. Findings indicate that the structural genes for the d-ribokinase and d-ribose permease map closely linked to the ara-leu region of the chromosome in contrast to their location in the isoleucine-valine region at 73.5 min in E. coli K12. Two polarity mutants, AB7 and AB36, were found to map at the left end of the d-ribokinase gene thus supporting the proposed d-ribokinase-d-ribose permease operon for the d-ribose catabolic enzymes in E. coli B/r.     

A mutated PtsG, the glucose transporter, allows uptake of D-ribose.

Mutations arose from an Escherichia coli strain defective in the high (Rbs/ribose) and low (Als/allose and Xyl/xylose) affinity D-ribose transporters, which allow cells to grow on D-ribose. Genetic tagging and mapping of the mutations revealed that two loci in the E. coli linkage map are involved in creating a novel ribose transport mechanism. One mutation was found in ptsG, the glucose-specific transporter of phosphoenolpyruvate:carbohydrate phosphotransferase system and the other in mlc, recently reported to be involved in the regulation of ptsG. Five different mutations in ptsG were characterized, whose growth on D-ribose medium was about 80% that of the high affinity system (Rbs+). Two of them were found in the predicted periplasmic loops, whereas three others are in the transmembrane region. Ribose uptakes in the mutants, competitively inhibited by D-glucose, D-xylose, or D-allose, were much lower than that of the high affinity transporter but higher than those of the Als and Xyl systems. Further analyses of the mutants revealed that the rbsK (ribokinase) and rbsD (function unknown) genes are involved in the ribose transport through PtsG, indicating that the phosphorylation of ribose is not mediated by PtsG and that some unknown metabolic function mediated by RbsD is required. It was also found that D-xylose, another sugar not involved in phosphorylation, was efficiently transported through the wild-type or mutant PtsG in mlc-negative background. The efficiencies of xylose and glucose transports are variable in the PtsG mutants, depending on their locations, either in the periplasm or in the membrane. In an extreme case of the transmembrane change (I283T), xylose transport is virtually abolished, indicating that the residue is directly involved in determining sugar specificity. We propose that there are at least two domains for substrate specificity in PtsG with slightly altered recognition properties.     

Catabolism of D-fructose and D-ribose by Pseudomonas doudoroffii. I. Physiological studies and mutant analysis.

Pseudomonas doudoroffii, a strict aerobe of marine origin, was able to utilize fructose and ribose but not glucose, gluconate, or other hexoses, pentoses, or sugar alcohols as sole sources of carbon and energy. Evidence was presented indicating that in this organism fructose was utilized via an inducible P-enolpyruvate: fructose phosphotransferase system (FPTS) which catalyzed the phosphorylation of fructose in the 1 position. The resulting fructose-1-P (F-1-P) was converted to fructose-1,6-P2 (FDP) by means of an inducible 1-P-fructokinase (1-PFK). The subsequent conversion of FDP to pyruvate involved enzymes of the Embden-Meyerhof pathway (EMP) which, with the exception of glyceraldehyde-3-P dehydrogenase (G3PDH), were constitutive. Two G3PDH activities were detected, one of which was inducible and NAD-dependent while the other was constitutive and NADP-dependent. Cell-free extracts of P. doudoroffii also contained enzymes of the methylglyoxal pathway (MGP) which converted dihydroxyacetone-P to pyruvate. The low specific activities of enzymes of this pathway as compared to the EMP suggested that the major route of FDP catabolism was via the latter pathway. 2. Ribose catabolism appeared to involve an inducible uptake system and an inducible ribokinase, the resulting ribose-5-P being converted to glyceraldehyde-3-P and fructose-6-P (F-6-P) by means of constitutive activities of the pentose-P pathway. The F-6-P formed as a result of these reactions was converted to FDP by means of a constitutive 6-P-fructokinase (6-PFK). Since no activity converting fructose or F-1-P to F-6-P could be detected in cell-free extracts of P. doudoroffii, the results suggested that fructose and ribose were catabolized via 1-PFK and 6-PFK, respectively, the two pathways converging at the level of FDP. Further evidence for this suggestion was obtained from a mutant which lacked an NAD-dependent G3PDH, accumulated FDP from both fructose and ribose, and was not able to grow on either of these compounds. 3. Ribose grown cells had increased amounts of the fructose uptake system and 1-PFK suggesting that a compound (or compounds) common to the catabolism of both fructose and ribose acted as the inducer(s) of these activities. Evidence was presented suggesting that the probable inducer(s) of 1-PFK and FPTS could be FDP, glyceraldehyde-3-P, or dihydroxyacetone-P. 4. A mutant unable to grow on fructose was characterized and found to lack FPTS while retaining 1-PFK and other enzyme activities of the EMP and MGP, indicating that a functional FPTS was essential for growth on fructose and suggesting that all or most of this sugar was catabolized via F-1-P.     

Inhibition of cell proliferation by D-ribose and deoxy-D-ribose

D-Ribose and deoxy-D-ribose inhibited DNA, RNA, and protein synthesis in a wide variety of cells (dividing and nondividing, normal and neoplastic, freely floating and substrate adhering, human and murine) at concentrations at which other monosaccharides have little or no effect. Inhibition was irreversible and proportional to the sugar concentration and time of contact. However, the first effects were seen only after 24 hr of incubation and progressed slowly to cell death. Whether the two sugars share the same mechanisms of action is not known. In any case, they deeply disturb metabolic processes in both dividing and nondividing cells.     

D-ribose inhibits DNA repair synthesis in human lymphocytes

D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.