Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Evidence for transketolase-like TKTL1 flux in CHO cells based on parallel labeling experiments and 13C-metabolic flux analysis

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-¹³C]glucose+[4,5,6-¹³C]glucose, [2-¹³C]glucose+[4,5,6-¹³C]glucose, and [3-¹³C]glucose+[4,5,6-¹³C]glucose and developed a new method to measure ¹³C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells.     

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.     

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.     

Strategies of isoprenoids production in engineered bacteria

Isoprenoids are the largest family of natural products, over 40 000 compounds have been described, which have been widely used in various fields. Currently, the isoprenoid products are mainly produced by natural extraction or chemical synthesis, however, limited yield and high cost is far behind the increasing need. Most bacteria synthesize the precursors of isoprenoids through the methylerythritol 4‐phosphate pathway, microbial synthesis of isoprenoids by fermentation becomes more attractive mainly in terms of environmental concern and renewable resources. In this review, the strategies of isoprenoid production in bacteria by synthetic biology are discussed. Introducing foreign genes associated with desired products made it possible to produce isoprenoids in bacteria. Furthermore, the yield of isoprenoids is increased by the strategies of overexpression of native or foreign genes, introducing heterologous mevalonate pathway, balancing of the precursors and inactivating the competing pathway, these methods were used separately or simultaneously.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Significance of the non-oxidative route of the pentose phosphate pathway for supplying carbon to the purine-nucleotide pathway in <Emphasis Type="Italic">Corynebacterium ammoniagenes</Emphasis>

To evaluate the strategy of supplying ribose 5-phosphate to the purine-nucleotide pathway exclusively via the nonoxidative route, the glucose 6-phosphate dehydrogenase gene zwf was disrupted in inosine- and 5′-xanthylic acid-producers of Corynebacterium ammoniagenes. In both producers, interruption of the oxidative route caused a decrease in production yields of about 50%. Attempts to increase the capacity of the nonoxidative route through overexpression of the transketolase or transaldolase gene in the zwf mutants led to no discernable effects on production, indicating that, in C. ammoniagenes, the nonoxidative route alone cannot provide sufficient ribose 5-phosphate for high-level production, although nonoxidative synthesis of the precursor is possible. Electronic Publication     

D-核糖生产菌的选育

将枯草芽胞杆菌通过紫外线诱变得到了莽草酸缺陷突变株,在２８株突变株中有１０株积累Ｄ－核糖。这些菌株均属戊糖磷酸途径的非氧化支路缺失突变株。对这些菌株的产核糖能力进行了验证、培养基中芳香族氨基酸的浓度影响Ｄ－核糖的积累     

Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26 μM of BCB for 90 min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB−: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10 min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB−: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB−). After fertilization, the zygotes produced from OBCB+ and OBCB− oocytes were further stained with BCB for 10 min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB−, OBCB−/ZBCB++, OBCB−/ZBCB+ and OBCB−/ZBCB−). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P < 0.05) than ZBCB treatment (ZBCB−; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB−: 28.8%) was less (P < 0.01) than single application of BCB test (ZBCB−: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%) were greater (P < 0.05) than that from C oocytes (25.7%), OBCB− oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P > 0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB−: 48.0%) was greater (P < 0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB−: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.     

产甲烷条件下岩溶湿地沉积物中古菌群落的变化规律

【背景】桂林会仙湿地位于喀斯特峰丛洼地和峰丛平原的过渡区,主要由岩溶地下河补给,水质呈现富钙偏碱特征,是一处典型的岩溶湿地环境,湿地沉积物以厌氧环境为主,独特的环境特征使之成为研究新型厌氧微生物的理想场所。氢型产甲烷菌和乙酸型产甲烷菌是环境中有机质厌氧降解的重要参与者,但目前会仙湿地产甲烷菌生理生态功能的研究十分有限。【目的】揭示乙酸盐营养型产甲烷条件和氢气营养型产甲烷条件下岩溶湿地环境古菌群落结构的变化规律和产甲烷菌的主要类群,探讨岩溶环境古菌的功能和相互关系。【方法】以会仙岩溶湿地沉积物为研究材料,分别以乙酸盐和氢气为唯一能源物进行富集培养,结合未添加任何能源底物的对照处理,动态监测产甲烷通量,分别构建了3个共包含146条古菌16S rRNA基因全长序列的文库,以及3个共包含138条甲基辅酶M还原酶基因mcr A基因序列的文库,通过构建系统发育树比较分析不同产甲烷底物培养条件下典型岩溶湿地古菌群落的变化规律。【结果】从乙酸型和氢型产甲烷条件培养物顶空气中都检测到了几乎等浓度的甲烷产生,甲烷八叠球菌是mcr A文库中主要的古菌类群,其中包含了Methanosarcina属古菌、Zoige Cluster I (ZC-I)和ANME-2d Cluster (AD)类群古菌,以及两个相对独立且不包含任何参考序列的分支KT-I和KT-II,可能代表产甲烷菌的新类群;经过两种产甲烷条件的富集培养后,ZC-I、AD和KT-II类群古菌的序列数占比有较为显著的增加(45%–155%);深古菌(Bathyarchaeota)是所有古菌16Sr RNA基因文库的优势类群,序列占比为88%–100%,其中MCG-11亚群最为丰富,占所有深古菌的84%,并且在乙酸盐产甲烷条件下增加了17%。【结论】会仙湿地沉积物中蕴涵着丰富的新型古菌序列,沉积物中主要的氢型产甲烷菌和乙酸型产甲烷菌都来自甲烷八叠球菌目,深古菌在岩溶环境和乙酸型产甲烷条件下可能都发挥着重要的作用。     

The Functional Significance of the Pentose Phosphate Pathway in Synaptosomes: Protection Against Peroxidative Damage by Catecholamines and Oxidants

Abstract: Catecholamines added in vitro in rat brain synaptosomes activate the decarboxylation of glucose radioactively labelled on carbon 1, suggesting an effective activation of the pentose phosphate pathway. Stimulation also occurred with phenazine methosulphate, reduced glutathione and hydrogen peroxide. The activation of the pentose phosphate pathway by 5-hydroxytryptamine, noradrenaline and dopamine is ascribed to the activation of monoamine oxidase, producing both the respective biogenic aldehyde and hydrogen peroxide. Evidence is presented that the further metabolism of the aldehyde by aldehyde reductase and the removal of hydrogen peroxide by glutathione peroxidase both release the limitation of N ADP⁺ availability for the pentose phosphate pathway by leading to the oxidation of NADPH. The relevance of the maintenance of reduced NADP⁺ on brain is discussed in relation to the metabolism of glutathione and to lipid peroxidation.     

Human Brain 6-Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.     

From peptidoglycan to glycoproteins: Common features of lipid-linked oligosaccharide biosynthesis

Abstract The peptidoglycan layer of bacterial cell walls is biosynthesised using a lipid carrier undecaprenyl phosphate to assemble and transport the MurNAc(GlcNAc)-pentapeptide precursor. Similar lipid-linked cycles are involved in the biosynthesis of other bacterial exopolysaccharides and eukaryotic asparagine-linked glycoproteins, the latter involving the structurally related dolichyl phosphate as a lipid carrier. Recent protein sequence data and common inhibitors of the bacterial and eukaryotic systems have revealed functional similarities between the two systems. Biological and physical studies on the lipid carriers themselves have provided clues to their role in oligosaccharide translocation, but have not revealed significant differences in function between undecaprenyl phosphate and dolichyl phosphate. The presence of dolichyl phosphate and a family of saturated isoprenoid lipids in Archaebacteria suggests a possible evolutionary link between the two systems.     

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge.     

Glucose is necessary for stabilization of hypoxia-inducible factor-1α under hypoxia: Contribution of the pentose phosphate pathway to this stabilization

In this study, we observed that low glucose or fructose reduces the increase in hypoxia-inducible factor-1α (HIF-1α) protein under hypoxic conditions. 6-Aminonicotinamide (6-AN), an inhibitor of the pentose phosphate pathway (PPP), also inhibited the increase of HIF-1α protein under hypoxic conditions, while the reduced protein levels of HIF-1α by low glucose were apparently recovered by the addition of MG-132 or NADPH. Moreover, siRNA for glucose-6-phosphate dehydrogenase, which produces NADPH, reduced the increase in HIF-1α protein. On the other hand, cobalt-induced expression of HIF-1α protein was not affected by low glucose or 6-AN under normoxic conditions. In conclusion, glucose metabolism through the PPP, but not in glycolysis, plays an important role in the stabilization of HIF-1α protein under hypoxic conditions.     

Phosphatases and phosphate affect the formation of glucose from pentoses in Escherichia coli

Metabolically engineered Escherichia coli MEC143 with deletions of the ptsG, manZ, glk, pfkA, and zwf genes converts pentoses such as arabinose and xylose into glucose, with the dephosphorylation of glucose‐6‐phosphate serving as the final step. To determine which phosphatase mediates this conversion, we examined glucose formation from pentoses in strains containing knockouts of six different phosphatases singly and in combination. Deletions of single phosphatases and combinations of multiple phosphatases did not eliminate the accumulation of glucose from xylose or arabinose. Overexpression of one phosphatase, haloacid dehalogenase‐like phosphatase 12 coded by the ybiV gene, increased glucose yield significantly from 0.26 to 0.30 g/g (xylose) and from 0.32 to 0.35 g/g (arabinose). Growing cells under phosphate‐limited steady‐state conditions increased the glucose yield to 0.39 g glucose/g xylose, but did not affect glucose yield from arabinose (0.31 g/g). No single phosphatase is exclusively responsible for the conversion of glucose‐6‐phosphate to glucose in E. coli MEC143. Phosphate‐limited conditions are indeed able to enhance glucose formation in some cases, with this effect likely influenced by the different phosphate demands when E. coli metabolizes different carbon sources.