D-Ribulose Production by a Thiamine-requiring Corynebacterium Species

The effect of thiamine on the D-ribulose production from gluconate by a thiamine-requiring Corynebacterium species was investigated. The D-ribulose production by the cells previously grown in a thiamine-deficient medium was higher than that by the cells grown in a thiamine-rich medium and supplementation of the thiamine-deficient cells with thiamine resulted in a significant depression of the D-ribulose production. Gluconokinase and NADP-linked phosphogluconate dehydrogenase were detected in the cell-free extract of this organism. Oxidation and anaerobic dissimilation of D-ribose 5-phosphate by the cell-free extract of the thiamine-deficient cells are reduced and the addition of thiamine pyrophosphate to the extract enhanced the catabolic activities for D-ribose 5-phosphate. These results suggest that the accumulation of D-ribulose by the thiamine-deficient cells is a consequence of a reduction of transketolase activity.     

Xylitol Production by a Corynebacterium Species

The washed cells of a gluconate-utilizing Corynebacterium strain grown in a gluconate- xylose medium produced xylitol from D-xylose in the presence of gluconate. The amount of xylitol was progressively increased with increasing gluconate concentration.

An extract of cells grown in the gluconate-xylose medium showed NADPH-dependent D-xylose reductase activity and NADP-dependent 6-phosphogluconate dehydrogenase activity.

These enzymes in the cell-free extract were purified by Sephadex G–100 gel filtration.

The reduction of D-xylose to xylitol was demonstrated by the coupling the D-xylose reductase activity to the 6-phosphogluconate dehydrogenase activity with NADP as a cofactor using the cell-free extract and the fractionated enzymes.     

Gluconate Metabolism in Escherichia coli

On the basis of information available in the literature, gluconate dissimilation in Escherichia coli is thought to occur via the hexose monophosphate pathway. Evidence is presented in this study that gluconate is catabolized in this organism via an inducible Entner-Doudoroff pathway. This evidence is based on chromatographic examination of end products produced from (14)C-labeled gluconate or glucose, distribution of (14)C in the carbon atoms of pyruvate formed from specifically labeled (14)C-glucose and (14)C-gluconate, and the ability of cell-free extracts to produce pyruvate from 6-phosphogluconate. Degradation of gluconate by an Entner-Doudoroff pathway occurred simultaneously with a glycolytic cleavage of glucose. A relationship between gluconate-induced, Entner-Doudoroff pathway activity and catabolism of glucose in Escherichia coli and other bacterial species is discussed.     

Tryptophol Metabolism by a Species of Arthrobacter

The stereochemistry of some dihydrofurano-isoflavones previously isolated from white lupin roots, or obtained following fungal metabolism of prenylated isoflavones, was investigated using CD spectroscopy. The osmate ester/pyridine complex of dextrorotatory lupinisoflavone A (1) exhibited a positive CD Cotton effect at 480 nm, indicating a side-structure configuration (S at C- 2″), opposite to that of natural rotenone (9), which afforded a negative Cotton effect at 474 nm (R- configuration at C-2′ on the side structure [ring E]). The stereochemistry of the laevorotatory luteone metabolite BC-1 (2) and lupinisoflavone D (4) (both ^-configuration at C-2″) was similarly determined after converting to the corresponding dehydrate (10) or trimethyl-dehydrate (1b, 10a).     

Mutations Affecting Gluconate Metabolism in Escherichia coli

A mutant of Escherichia coli K-12 that does not ferment gluconate on fermentation plates was isolated and characterized. This mutant, designated M2, shows a long lag for growth on gluconate mineral medium and somewhat reduced levels of high-affinity transport, gluconokinase, and gluconate-6-P dehydrase activities in the log phase of growth. The mutation involved is near malA. Deletion mutants in which malA region was affected were also studied. They were found to affect the function of different genes involved in gluconate metabolism.     

Vanillate Metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.     

Metabolism of l-Malate and d-Malate by a Species of Pseudomonas

Extracts of a fluorescent species of Pseudomonas grown with m-cresol, degrade gentisic acid without isomerization of the ring-fission compound, maleylpyruvate, to give eventually d-malate and pyruvate. d-Malate is also a growth substrate. l-Malate but not d-malate is oxidized by a particulate enzyme not requiring nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). NAD- or NADP-linked malate dehydrogenases are absent but cells contain an NADP-dependent l-malic enzyme. Exposure of cells to exogenous d-malate induces an NAD-dependent d-malic enzyme, not present when d-malate is formed endogenously. Succinate- or m-cresol-grown cells, containing no d-malic enzyme, rapidly oxidize d-malate in the presence of chloramphenicol at a concentration suffient to inhibit protein synthesis. An NADP-dependent cell-free system, prepared from succinate-grown cells which oxidized d-malate, is described.     

Carbohydrate Metabolism Mutants of a Bacillus Species

Mutants which could not utilize d-gluconate, l-arabinose, sorbitol, pyruvate or l-glutamate as a sole carbon source and which required shikimic acid for their growth were isolated. Characterization of these mutants by the patterns of carbohydrate utilization revealed that various kinds of carbohydrate metabolism mutants including those of the non-oxidative limb of the pentose phosphate pathway were isolated.

Ability of inosine formation of these mutants and transformants from them was investigated. Consequently, slightly improved strains were found among transformants in comparison with the parent strain.     

Metabolism of Dibenzo-p-Dioxin and Chlorinated Dibenzo-p- Dioxins by a Beijerinckia Species

Whole cells of the parent strain of Beijerinckia, grown with succinate and biphenyl, oxidized dibenzo-p-dioxin and several chlorinated dioxins. The rate of oxidation of the chlorinated dibenzo-p-dioxins decreased with an increasing degree of chlorine substitution. A mutant strain (B8/36) of Beijerinckia oxidized dibenzo-p-dioxin to cis-1,2-dihydroxy-1,2-dihydrodibenzo-p-dioxin. The mutant organism also oxidized two monochlorinated dibenzo-p-dioxins to cis-dihydrodiols. No metabolites were detected from two dichlorinated dibenzo-p-dioxins. Growth of the parent strain of Beijerinckia on succinate was inhibited after 4 h when 0.05% dibenzo-p-dioxin was present in the culture medium. Resting cell suspensions of the parent organism, previously grown with succinate and biphenyl, oxidized dibenzo-p-dioxin to a compound identified as 1,2-dihydroxydibenzo-p-dioxin. Further degradation of this metabolite was not detected, as the compound was found to be a potent mixed-type inhibitor of two ring-fission oxygenases present in this organism.     

Metabolism of Nitrilotriacetate by Cells of Pseudomonas Species

A Pseudomonas species was isolated from soil which could degrade nitrilotriacetate (NTA) to CO(2), H(2)O, NH(3), and cellular constituents without the accumulation of significant quantities of intermediates either in the presence or absence of several inhibitors. After extensive gas chromatography analysis, small quantities of aspartate, glycine, and aconitate were the only detectable compounds to accumulate during NTA degradation, and these compounds were not excreted from the cells. Manometric studies indicated that iminodiacetate, glycine, and glyoxylate are possible intermediates, whereas N-methyliminodiacetate, sarcosine, and acetate are not. The data are consistent with an oxidative cleavage of the C-N bond of NTA as the initial degradation step.     

Metabolism of Linear Alcohols with Various Chain Lengths by a Pseudomonas Species

Pseudomonas C12B grew on and oxidized linear primary alcohols with even- and odd-numbered carbon chains ranging from C(2) to C(11). Cell-free extracts of the bacteria contained a nicotinamide adenine dinucleotide-linked dehydrogenase(s) active with these alcohols and with branched primary and linear secondary alcohols as well. Analysis by gas-liquid chromatography of hexane extracts of filtrates of cultures containing mixtures of even-carbon numbered alcohols from C(10) to C(18) revealed that decanol was rapidly utilized, whereas the remainder were slowly dissimilated up to 19 hr and then were rapidly degraded in the next few hours of culture. The validity for these studies of (i) steam distillation as a method for collecting the alcohols from cultures, and (ii) quantitative estimation by gas-liquid chromatographic comparison with an added internal marker, was established. Steam distillation and gas-liquid chromatography were then used to show that failure to demonstrate stoichiometry of sulfate and dodecanol in the alkyl sulfatase reaction in a previous study resulted from contamination of the commercial "Dodecyl Sodium Sulfate, 95%" used with decyl, undecyl, and tetradecyl sulfates.     

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [¹³C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO₂, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.     

Glucose and Gluconate Metabolism in a Mutant of Escherichia coli Lacking Gluconate-6-phosphate Dehydrase

A mutant lacking gluconate-6-phosphate dehydrase (the first enzyme of the Entner-Doudoroff pathway) was isolated after ethyl methane sulfonate mutagenesis of Escherichia coli. Other enzymes of gluconate metabolism (gluconokinase, gluconate-6-phosphate dehydrogenase, and 2-keto-3-deoxygluconate-6-phosphate aldolase) were present in the mutant. When the mutant was grown on gluconate-1-(14)C, alanine isolated from protein was unlabeled, showing that the dehydrase was absent in vivo and that the sole pathway of gluconate metabolism in the mutant was the hexose monophosphate shunt. The mutant grew on gluconate with a doubling time of 155 min, compared with the parent strain's 56 min. On glucose and fructose it grew with normal doubling times. Thus, in E. coli, the Entner-Doudoroff pathway is used for gluconate metabolism but not for glucose metabolism.     

Glucose and Gluconate Metabolism in an Escherichia coli Mutant Lacking Phosphoglucose Isomerase

A single gene mutant lacking phosphoglucose isomerase (pgi) was selected after ethyl methane sulfonate mutagenesis of Escherichia coli strain K-10. Enzyme assays revealed no pgi activity in the mutant, whereas levels of glucokinase, glucose-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrogenase were similar in parent and mutant. The amount of glucose released by acid hydrolysis of the mutant cells after growth on gluconate was less than 2% that released from parent cells; when grown in the presence of glucose, mutant and parent cells contained the same amount of glucose residues. The mutant grew on glucose one-third as fast as the parent; it also grew much slower than the parent on galactose, maltose, and lactose. On fructose, gluconate, and other carbon sources, growth was almost normal. In both parent and mutant, gluconokinase and gluconate-6-phosphate dehydrase were present during growth on gluconate but not during growth on glucose. Assay and degradation of alanine from protein hydrolysates after growth on glucose-1-(14)C and gluconate-1-(14)C showed that in the parent strain glucose was metabolized by the glycolytic path and the hexose monophosphate shunt. Gluconate was metabolized by the Entner-Doudoroff path and the hexose monophosphate shunt. The mutant used glucose chiefly by the shunt, but may also have used the Entner-Doudoroff path to a limited extent.     

A Species of Corynebacterium Isolated from Fermenting Cassava Roots

S ummary : A species of Corynebacterium not described in the 7th edition of Bergey's Manual has been isolated from grated cassava ( Manihot utilissima ) root allowed to ferment for some days during the preparation of 'gari', a farinaceous food eaten in Nigeria. The organism ferments starch, dextrose, maltose, sucrose, salicin, xylose and arabinose with the production of acid only, and produces bright yellow colonies of a characteristic form on litmus-lactose agar. It has been suggested that this organism, if accorded specific status, should be named Corynebacterium manihot.     

Link between Phosphate Starvation and Glycogen Metabolism in Corynebacterium glutamicum,Revealed by Metabolomics

In this study, we analyzed the influence of phosphate (P_i) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under P_i limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under P_i excess, the glycogen level in P_i-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under P_i excess, did so under P_i limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under P_i limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited P_i concentrations.Phosphorus is an essential nutrient for all cells and is required for, e.g., the biosynthesis of nucleotides, NAD(P)H, DNA, and RNA but also for the regulation of protein activity by phosphorylation of histidine, aspartate, serine, threonine, or tyrosine residues. A common phosphorus source is inorganic phosphate (P_i), and cells have developed mechanisms for the acquisition, assimilation, and storage of P_i. When P_i becomes limiting, many bacteria induce the synthesis of proteins that enable them to capture the residual P_i resources more efficiently and to make alternative phosphorus sources accessible. The corresponding genes are collectively named P_i starvation-inducible genes, or psi genes. The P_i starvation response, and in particular its regulation, has been most carefully studied in Escherichia coli (45) and Bacillus subtilis (14).We recently started to characterize the P_i starvation response in Corynebacterium glutamicum, a Gram-positive soil bacterium used industrially for the production of more than two millions tons of amino acids per year, mainly l-glutamate and l-lysine (12). An overview of the biology, genetics, physiology, and application of C. glutamicum can be found in two recent monographs (3, 6). Phosphorus constitutes 1.5% to 2.1% of the cell dry weight of C. glutamicum (24), part of which can be present as polyphosphate (22, 29). Several of the enzymes involved in polyphosphate metabolism have been characterized recently, such as a class II polyphosphate kinase (28), the exopolyphosphatases Ppx1 and Ppx2 (26), a polyphosphate/ATP-dependent glucokinase (25), and a polyphosphate/ATP-dependent NAD⁺ kinase (27). The P_i starvation stimulon of C. glutamicum was determined using whole-genome DNA microarrays (15). Comparison of the mRNA profiles before and at different times after a shift from P_i excess to P_i starvation led to the identification of a group of genes that are presumably required to cope with limited P_i supply. This group includes the following: the pstSCAB operon, encoding an ABC transporter for high-affinity P_i uptake; the ugpAEBC operon, encoding an ABC transporter for uptake of glycerol 3-phosphate; glpQ1, encoding a glycerophosphoryl diester phosphodiesterase; ushA, encoding a secreted enzyme with UDP-sugar hydrolase and 5′-nucleotidase activities (33); nucH, encoding a putative secreted nuclease which possibly plays a role in liberating P_i from extracellular nucleic acids; phoC (NCgl2959/cg3393), which may encode a cell wall-associated phosphatase (46); phoH1, encoding an ATPase of unknown function; and the pctABCD operon, encoding an ABC transport system which might be involved in the uptake of a yet-unknown phosphorus-containing compound (15). C. glutamicum lacks homologs of genes for phosphonate degradation, as well as the capability to utilize phosphonates as P sources (15).In most bacteria analyzed in this respect, the P_i starvation response is controlled by two-component signal transduction systems, e.g., the PhoBR system in E. coli (13) and the PhoPR system in B. subtilis (14). Our previous studies revealed that in C. glutamicum, a two-component system composed of the sensor kinase PhoS and the response regulator PhoR is involved in the activation of phosphate starvation-inducible genes (21). Studies with purified proteins showed that phosphorylation by PhoS increased the DNA-binding affinity of PhoR, which bound to many of the P_i starvation-inducible genes, but with different affinities (34).The study reported here was initiated by the question how the metabolism of C. glutamicum responds to P_i limitation. Our results reveal a link between P_i limitation and glycogen metabolism, which was also used for metabolic simulations based on a genome-wide metabolic model.     

Biosynthesis of Riboflavine in Corynebacterium Species: the Purine Precursor

Corynebacterium species lacks the ability to convert either xanthine or guanine to adenine. This defect and the use of the purine nucleoside antibiotic decoyinine, which blocks the conversion of xanthosine monophosphate --> guanosine monophosphate, permit an experimental design in which the interconversion of purines is largely prevented. Cultures of this organism were grown in the presence of decoyinine and various purine supplements. Data obtained by comparing the radioactivity incorporated from guanine-2-(14)C or xanthine-2-(14)C into bacterial guanine, xanthine, and riboflavine indicate that guanine or a close derivative of guanine is the purine precursor of riboflavine.