Ribose biosynthesis in methanogenic bacteria

NMR spectroscopy was used to determine the labeling patterns of the ribose moieties of ribonucleosides purified from Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii labeled with ¹³C-precursors. In most methanogens tested ribose was labeled in a manner consistent with the operation of the oxidative branch of the pentose phosphate pathway. In contrast, transaldolase and transketolase reactions typical of a partial nonoxidative pentose phosphate pathway are hypothesized to explain the different labeling patterns and enrichments of carbon atoms observed in the ribose moiety of Methanococcus voltae. The source of erythrose 4-phosphate needed for the transaldolase reaction proposed in Methanococcus voltae, and for biosynthesis of aromatic amino acids in methanogenic bacteria in general, was assessed. Phenylalanine carbon atom C-7 was labeled by [1-¹³C]pyruvate in Methanospirillum hungatei, Methanococcus voltae, and Methanococcus jannaschii, the only methanogens which incorporated sufficient label from pyruvate for testing. Reductive carboxylation of a triose precursor (derived from pyruvate) to synthesize erythrose 4-phosphate is consistent with the labeling patterns observed in phenylalanine and ribose.Abbreviation TCA Tricarboxylic acid Issued as NRCC Publication No. 37382     

Purine and pyrimidine biosynthesis in higher plants

Purine and pyrimidine nucleotides have important functions in a multitude of biochemical and developmental processes during the life cycle of a plant. In higher plants the processes of nucleotide metabolism are poorly understood, but it is in principle accepted that nucleotides are essential constituents of fundamental biological functions. Despite of its significance, higher plant nucleotide metabolism has been poorly explored during the last 10–20 years (Suzuki and Takahashi 1977, Schubert 1986, Wagner and Backer 1992). But considerable progress was made on purine biosynthesis in nodules of ureide producing tropical legumes, where IMP-synthesis plays a dominant role in primary nitrogen metabolism (Atkins and Smith 2000, Smith and Atkins 2002). Besides these studies on tropical legumes, this review emphasises on progress made in analysing the function in planta of genes involved in purine and pyrimidine biosynthesis and their impact on metabolism and development.     

Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani

Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.     

Isolation and characterization of methanogenic bacteria from rice paddies

Abstract Enrichment cultures for H₂-CO₂, methanol- or acetate-utilizing methanogens were prepared from two rice field soil samples. All the cultures except one acetate enrichment showed significant methane production. Pure cultures of Methanobacterium - and Methanosarcina -like organisms were isolated from H₂-CO₂ and methanol enrichment cultures, respectively, and were characterized for various nutritional and growth conditions. The organisms had an optimal pH range of 6.4–6.6 and a temperature optimum of 37°C. The Methanobacterium isolates were able to utilize H₂-CO₂ but no other substrates as sole energy source, while the Methanosarcina isolates were able to utilize methanol, methylamines or H₂-CO₂ as sole energy sources. Both Methanobacterium isolates and one isolate of Methanosarcina were able to use dinitrogen as the sole source of nitrogen for growth. The isolates used several sulfur compounds as sole sources of sulfur.     

Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy

Purine salvage is an essential function for all obligate parasitic protozoa studied to date and most are also capable of efficient uptake of preformed pyrimidines. Much progress has been made in the identification and characterisation of protozoan purine and pyrimidine transporters. While the genes encoding protozoan or metazoan pyrimidine transporters have yet to be identified, numerous purine transporters have now been cloned. All protozoan purine transporter-encoding genes characterised to date have been of the Equilibrative Nucleoside Transporter family conserved in a great variety of eukaryote organisms. However, these protozoan transporters have been shown to be sufficiently different from mammalian transporters to mediate selective uptake of therapeutic agents. Recent studies are increasingly addressing the structure and substrate recognition mechanisms of these vital transport proteins.     

Pyrimidine and Purine Analogues,Effects on Cell Cycle Regulation and the Role of Cell Cycle Inhibitors to Enhance Their Cytotoxicity

In anti-cancer treatment, deoxynucleoside analogues are widely used in combination chemotherapy. Improvement can be achieved by rational design of novel combinations with cell cycle inhibitors. These compounds inhibit protein kinases, preventing the cell cycle from continuing when affected by deoxynucleoside analogs. The efficacy is dependent on the site of cell cycle inhibition, whether multiple cyclin-dependent kinases are inhibited and whether the inhibitors should be given before or after the deoxynucleoside analogs. The action of cell cycle inhibition in vivo may be limited by unfavorable pharmacokinetics. Preclinical and clinical studies will be discussed, aiming to design improved future strategies.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

Rhizobial purine and pyrimidine auxotrophs: nutrient supplementation,genetic analysis,and the symbiotic requirement for the novo purine biosynthesis

Previously described Rhizobium leguminosarum bv. phaseoli mutants elicit nodules on bean without infection thread formation. These mutants were shown to be purine or, in one case, pyrimidine auxotrophs. Each of the seven purine auxotrophs grew normally when supplied the penultimate precursor of inosine, 5-aminoimidazole-4-carboxamide riboside. Four seemed blocked early in the purine pathway, because they were also thiamine auxotrophs. Reversion analysis and genetic complementation using cloned wild-type DNA showed that in each mutant a single mutation was responsible for both the symbiotic defect and purine or pyrimidine auxotrophy. The mutations were mapped to five dispersed chromosomal locations. The previously reported weak Calcofluor staining of these mutants on minimal agar appeared to be caused by partial growth on contaminating nutrients in the agar, rather than deficient exopolysaccharide production. Nodulation by the mutants was not enhanced by supplying purine or pyrimidine compounds exogenously. Furthermore, with or without added purine, the purine auxotrophs grew in the root environment as well as the wild type. However, nodulation by the purine auxotrophs was enhanced greatly in the presence of 5-aminoimidazole-4-carboxamide riboside. The results suggest that undiminished metabolic flow through de novo purine biosynthesis, or a particular intermediate in the pathway, is essential in early symbiotic interactions.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

Modeling allosteric regulation of de novo pyrimidine biosynthesis in Escherichia coli

With the emergence of multifaceted bioinformatics-derived data, it is becoming possible to merge biochemical and physiological information to develop a new level of understanding of the metabolic complexity of the cell. The biosynthetic pathway of de novo pyrimidine nucleotide metabolism is an essential capability of all free-living cells, and it occupies a pivotal position relative to metabolic processes that are involved in the macromolecular synthesis of DNA, RNA and proteins, as well as energy production and cell division. This regulatory network in all enteric bacteria involves genetic, allosteric, and physiological control systems that need to be integrated into a coordinated set of metabolic checks and balances. Allosterically regulated pathways constitute an exciting and challenging biosynthetic system to be approached from a mathematical perspective. However, to date, a mathematical model quantifying the contribution of allostery in controlling the dynamics of metabolic pathways has not been proposed. In this study, a direct, rigorous mathematical model of the de novo biosynthesis of pyrimidine nucleotides is presented. We corroborate the simulations with experimental data available in the literature and validate it with derepression experiments done in our laboratory. The model is able to faithfully represent the dynamic changes in the intracellular nucleotide pools that occur during metabolic transitions of the de novo pyrimidine biosynthetic pathway and represents a step forward in understanding the role of allosteric regulation in metabolic control.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

嘌呤核苷及其衍生物的代谢工程

嘌呤核苷及其衍生物被广泛应用于食品和医药领域。利用诱变筛选技术可以获得嘌呤核苷类产品的工业生产菌株,但往往耗时,效率低,而且获得的某些高产菌株还存在不稳定的缺陷。菌株代谢调控与生理生化的研究为代谢工程优化嘌呤核苷类产品的合成提供了理论基础,利用代谢工程改造菌株合成嘌呤核苷也引起了研究人员的关注。系统地介绍了微生物嘌呤生物合成途径及其调控机制,综述了嘌呤核苷类产品及其衍生物的代谢工程研究进展,最后讨论了利用代谢工程改造菌株合成这些产品面临的问题及今后的研究方向。     

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M _n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 10⁶ and 2.5 × 10⁶ g mol⁻¹, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.     

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 μmol/l and 3–6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at −70°C. Plasma samples can be stored at −20°C for upt to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.     

Nucleic acid economy in bacteria infected with bacteriophage T2. I. Purine and pyrimidine composition

1. The phosphorus content per infective particle of isolated bacteriophage T2 has been redetermined. It does not exceed 1.8 to 2.2 x 10^–11 µg. The equivalent amount of DNA has been defined in terms of several analytical methods and taken as a unit of measurement of intrabacterial DNA. 2. The DNA of E. coli contains guanine, adenine, cytosine, and thymine in approximately equal amounts, but no hydroxymethylcytosine. One bacterial cell contains 40 to 150 units of DNA, depending on the conditions of growth and the method of measurement. 3. The DNA of phage T2 (one unit per particle by definition) contains guanine, 5-hydroxymethylcytosine, and relatively large amounts of adenine and thymine, but no cytosine. 4. Infected bacteria contain DNA of a composition that varies systematically during the course of viral growth. At all times it resembles a mixture of bacterial and viral DNA. 5. The characteristic bacterial DNA is decomposed after infection, measuring about one-third its initial amount at 20 minutes. The characteristic viral DNA increases in amount, after a short delay, reaching a level of 100 to 400 units per bacterium 30 minutes after infection. At 10 minutes after infection, the two kinds of DNA are approximately equal in amount. 6. The characteristic viral DNA present in infected cells exists in two forms, one consisting of infective particles and one not. The portion not contained in infective particles builds up to 40 to 80 units per cell during the first 10 minutes after infection and afterwards remains roughly constant in amount. Infective particles begin to appear at 10 minutes and account for all or most of the increase thereafter.     

A genetic and dietary study of the physiology of pyrimidine synthesis in Drosophila melanogaster

A combination of genetic and dietary manipulations have been utilized to investigate the physiology of pyrimidine metabolism throughout the Drosophila life cycle. We present evidence that the dietary sources of pyrimidines ingested at the larval stage are sufficient for all subsequent stages of the life cycle, except the process of oögenesis in adult females where a maternal supply of endogenously synthesized pyrimidines is normally required. Deprivation of dietary and de novo synthesis does not affect adult longevity; indicating that with the exception of oögenesis, all normal functions are carried out by recycling pyrimidines produced or ingested at the larval stage.In an enzymatic analysis, we have determined that Drosophila does not adjust for pyrimidine dietary deficiencies by significantly altering the level of synthesis of two tested enzymes encoded by the rudimentary locus, even though the pyrimidine deficiency is a rate limiting step in the completion of development.