Thermal Synthesis of Nucleoside H-Phosphonates Under Mild Conditions

Nucleosides react rapidly with ammonium phosphite ((NH₄)₂HPO₃) at 60 °C to produce good yields of nucleoside-5-phosphite monoesters within 24 h. Under the same conditions, ammonium phosphate is unreactive, producing low yields of nucleotide only after extended reactions. These results confirm earlier suggestions that nucleoside H-phosphonates and their possible condensation products may have been produced on the primitive earth more easily than nucleotides.     

Life based on phosphite: a genome-guided analysis of Desulfotignum phosphitoxidans

Background

The Delta-Proteobacterium Desulfotignum phosphitoxidans is a type strain of the genus Desulfotignum, which comprises to date only three species together with D. balticum and D. toluenicum. D. phosphitoxidans oxidizes phosphite to phosphate as its only source of electrons, with either sulfate or CO₂ as electron acceptor to gain its metabolic energy, which is of exclusive interest. Sequencing of the genome of this bacterium was undertaken to elucidate the genomic basis of this so far unique type of energy metabolism.

Results

The genome contains 4,998,761 base pairs and 4646 genes of which 3609 were assigned to a function, and 1037 are without function prediction. Metabolic reconstruction revealed that most biosynthetic pathways of Gram negative, autotrophic sulfate reducers were present. Autotrophic CO₂ assimilation proceeds through the Wood-Ljungdahl pathway. Additionally, we have found and confirmed the ability of the strain to couple phosphite oxidation to dissimilatory nitrate reduction to ammonia, which in itself is a new type of energy metabolism. Surprisingly, only two pathways for uptake, assimilation and utilization of inorganic and organic phosphonates were found in the genome. The unique for D. phosphitoxidans Ptx-Ptd cluster is involved in inorganic phosphite oxidation and an atypical C-P lyase-coding cluster (Phn) is involved in utilization of organophosphonates.

Conclusions

We present the whole genome sequence of the first bacterium able to gain metabolic energy via phosphite oxidation. The data obtained provide initial information on the composition and architecture of the phosphite–utilizing and energy-transducing systems needed to live with phosphite as an unusual electron donor.     

一种基于亚磷酸盐及其脱氢酶的植物磷利用和杂草控制系统的建立

磷是植物生长发育所必需的大量营养元素之一。土壤中存在大量的正磷酸盐 (Pi),但由于土壤化学和微生物转化使得土壤可利用磷的浓度并不高。土壤缺磷以及杂草的抗除草剂能力已成为当前农业可持续发展的重要限制因素,所以提高植物对土壤磷的吸收利用能力或寻求可替代正磷酸盐的磷肥以及开发新型杂草控制系统已成为亟待解决的问题。自然界中亚磷酸盐 (Phi) 是含量仅次于正磷酸盐的磷源,但仅在某些细菌中能被专一性的亚磷酸盐脱氢酶 (PTDH) 氧化利用,对植物的生长发育则具有抑制作用。利用这一特性,将从土壤宏基因组中直接扩增到的假单胞菌PTDH基因PsPtx通过农杆菌侵染法转入烟草中,并通过RT-PCR、垂直板幼苗生长、显性标记和生长竞争实验分析PsPtx转基因烟草的基因表达以及在Phi胁迫条件下的特性。结果显示,PsPtx在其转基因植株的根茎叶组织中都有几乎相同水平的表达;PsPtx转基因烟草不但能解除Phi对植物的毒害作用,并将它氧化成可用的Pi作为生长发育所需的磷源,而且在Phi胁迫条件下较野生型烟草有相当明显的生长竞争优势;另外PsPtx还具备成为植物遗传转化显性选择标记的优良特质。因此,PsPtx基因编码的亚磷酸盐脱氢酶可用于开发一种基于亚磷酸盐为磷肥和除草剂的植物磷利用和杂草控制系统,为当前农作物转基因研究存在的一些重大问题提供一个有效解决方案。     

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large‐scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods.     

Biodegradation of dibutyl phosphite by Sphingobium sp. AMGD5 isolated from aerobic granular biomass

In the present study, cultivation of aerobic granular biomass capable of biodegradation of dibutyl phosphite, an organophosphite, and isolation of dibutyl phosphite degrading bacterial strains, are reported for the first time. The strain AMGD5, identified as Sphingobium sp., based on 16S rRNA sequencing, degraded dibutyl phosphite efficiently and utilised it as the sole source of carbon and phosphorus. Microbial degradation of dibutyl phosphite caused a significant decrease in medium pH, leading to cessation of growth and further degradation of dibutyl phosphite. Under buffered conditions, complete degradation of up to 3 mM of dibutyl phosphite was achieved within 60 h. The strain showed almost similar growth pattern when either phosphite or dibutyl phosphite was used as the phosphorous source. A 4-fold enhancement in phosphatase activity was evident in dibutyl phosphite fed cells, implying their role in dibutyl phosphite degradation. Sphingobium sp. AMGD5 can be a potential candidate for bioremediation of dibutyl phosphite contaminated waters or sites.     

Utilization of 1,3-Dioxolanes in the Synthesis of α-branched Alkyl and Aryl 9-[2-(Phosphonomethoxy)Ethyl]Purines and Study of the Influence of α-branched Substitution for Potential Biological Activity

Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.     

A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae

Microalgal cultivation that takes advantage of solar energy is one of the most cost‐effective systems for the biotechnological production of biofuels, and a range of high value products, including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors, the control of contamination requires the sterilization of the media, containers and all materials, which increases the cost of production, whereas open pond systems severely limits the number of species that can be cultivated under extreme environmental conditions to prevent contaminations. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants.     

Synthesis, characterization of a new open-framework gallium phosphite containing 5,6-fold coordinate gallium atoms

A new open-framework gallium phosphite Ga₅(OH)₂(HPO₃)₈(C₄N₃H₁₆) · 2H₂O (1) is synthesized solvothermally using diethylenetriamine (DETA) as template from a mixed solvent system in which ethylene glycol (EG) was used as the co-solvent. The as-synthesized product is characterized by single crystal X-ray diffraction, powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis (TGA), ICP-AES and elemental analyses. The three-dimensional open-framework of compound 1 is constructed from two novel secondary building units (SBUs), Ga₂O₇(OH)(HPO₃) three-membered ring (3R) and Ga(HPO₃)₆ tooth-wheel type unit. These two novel secondary building units are first found in gallium phosphate/phosphite, which lead to form the one-dimensional 8-membered ring channels along the a-axis. Moreover, it is noted that compound 1 is the first gallium phosphite containing 5-fold coordinate gallium atom.     

Synthesis of sterically hindered peptide analogs using diphenyl phosphite as the coupling reagent

Some model sterically hindered peptide analogs were synthesized with various yields using diphenyl phosphite as the coupling reagent under mild conditions. The experimental procedure is straightforward and the products are easily isolated. This method may be convenient and efficient for the synthesis of hindered peptides.     

THE EFFECTS OF PHOSPHITE ON PHOSPHATE STARVATION RESPONSES OF ULVA LACTUCA (ULVALES,CHLOROPHYTA)1

Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH₂PO₄) or Pi‐sufficient (100 μM NaH₂PO₄) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH₂PO₄ decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The K_m value of Pi uptake rate was decreased by incubation with 1 μM NaH₂PO₄ and the decrease in K_m value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions.     

Environmentally benign beta-stereoselective glycosidations of glycosyl phosphites using a reusable heterogeneous solid acid, montmorillonite K-10

Environmentally benign and stereoselective beta-glycosidations of glycopyranosyl phosphites and alcohols using a reusable heterogeneous solid acid, montmorillonite K-10, as an activator have been developed. By these glycosidations, beta-gluco-, 2-deoxy-beta-gluco-, and beta-mannopyranosides were selectively produced in good to high yields.     

Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.     

Study of physio-biochemical responses elicited by potassium phosphite in downy mildew-infected cucumber plants

Potassium phosphite (KPhi) is widely used as a resistance inducer to protect plants against fungal pathogens. In the present study, the effect of KPhi on the activation of defence-related enzymes and biochemicals in Pseudoperonospora cubensis-challenged cucumber plants was investigated. Cucumber plants were treated with KPhi before or after inoculation with P. cubensis and leaf samples were collected at different time courses for physiological and biochemical assessments. Results revealed that the activity of reactive oxygen species (ROS)-scavenging enzymes like catalase, guaiacol peroxidase, superoxide dismutase and ascorbate peroxidase as well as proline and total carbohydrates contents were significantly increased by KPhi application, while hydrogen peroxide (H₂O₂) concentration, as a disease damage indicator was reduced. The maximum activity of ROS-scavenging system was achieved 3–4 days after KPhi application. These findings suggest that KPhi application prior to pathogen infection efficiently triggers plant defence responses which may reduce the disease severity.     

The role of potassium phosphite in chlorophyll fluorescence and photosynthetic parameters of downy mildew-challenged cucumber Cucumis sativus plants

The effect of potassium phosphite (KPhi) on the photosynthetic parameters of cucumber plants inoculated with Pseudoperonospora cubensis was investigated in the present study. Cucumber plants were treated with KPhi before or after inoculation with P. cubensis and leaf samples were collected at different time courses for assessments. Results showed that in pathogen-inoculated plants Fv/Fm was decreased up to 3%. The rate of quantum photosynthetic was also decreased significantly in inoculated plants. Downy mildew led to a decrease in chlorophyll amount which in turn reduced the efficiency of photosystem II. In the KPhi-treated leaves, chlorophyll a and b decreased by 72% and 68%, respectively. Remarkable reduction in the efficiency of photosystem II as well as increased lipid membrane disruption, led to increased lipid peroxidation rate of the membranes up to 52%. The results of this study indicate the mitigating role of potassium phosphite in reducing the adverse effects of pathogen and maintaining the photosynthetic apparatus efficiency in cucumber plants.     

Potential for phosphite and phosphonate utilization by Prochlorococcus

Phosphonates (Pn) are diverse organic phosphorus (P) compounds containing C–P bonds and comprise up to 25% of the high-molecular weight dissolved organic P pool in the open ocean. Pn bioavailability was suggested to influence markedly bacterial primary production in low-P areas. Using metagenomic data from the Global Ocean Sampling expedition, we show that the main potential microbial contributor in Pn utilization in oceanic surface water is the globally important marine primary producer Prochlorococcus. Moreover, a number of Prochlorococcus strains contain two distinct putative Pn uptake operons coding for ABC-type Pn transporters. On the basis of microcalorimetric measurements, we find that each of the two different putative Pn-binding protein (PhnD) homologs transcribed from these operons possesses different Pn- as well as inorganic phosphite-binding specificities. Our results suggest that Prochlorococcus adapt to low-P environments by increasing the number of Pn transporters with different specificities towards phosphite and different Pns.     

Environmentally benign and stereoselective formation of beta-mannosidic linkages utilizing 2,3-di-O-benzyl-4,6-O-benzylidene-protected mannopyranosyl phosphite and montmorillonite K-10

An environmentally benign and stereoselective beta-mannopyranosylation has been developed employing 4,6-O-benzylidene-protected mannopyranosyl diethyl phosphite as a glycosyl donor and montmorillonite K-10 as an activator.     

Phosphate and phosphite have a differential impact on the proteome and phosphoproteome of Arabidopsis suspension cell cultures

Phosphorus absorbed in the form of phosphate (H₂PO₄⁻) is an essential but limiting macronutrient for plant growth and agricultural productivity. A comprehensive understanding of how plants respond to phosphate starvation is essential for the development of more phosphate-efficient crops. Here we employed label-free proteomics and phosphoproteomics to quantify protein-level responses to 48 h of phosphate versus phosphite (H₂PO₃⁻) resupply to phosphate-deprived Arabidopsis thaliana suspension cells. Phosphite is similarly sensed, taken up and transported by plant cells as phosphate, but cannot be metabolized or used as a nutrient. Phosphite is thus a useful tool for differentiating between non-specific processes related to phosphate sensing and transport and specific responses to phosphorus nutrition. We found that responses to phosphate versus phosphite resupply occurred mainly at the level of protein phosphorylation, complemented by limited changes in protein abundance, primarily in protein translation, phosphate transport and scavenging, and central metabolism proteins. Altered phosphorylation of proteins involved in core processes such as translation, RNA splicing and kinase signaling was especially important. We also found differential phosphorylation in response to phosphate and phosphite in 69 proteins, including splicing factors, translation factors, the PHT1;4 phosphate transporter and the HAT1 histone acetyltransferase – potential phospho-switches signaling changes in phosphorus nutrition. Our study illuminates several new aspects of the phosphate starvation response and identifies important targets for further investigation and potential crop improvement.     

The role of 4,4′-trimethylene-dipyridine flexibility in the construction of hybrid networks templated on aromatic alcohols

A series of zinc phosphite hybrid structures with trimethylene dipyridine as the linking ligand and templated on the aromatic alcohols, hydroquinone, 1,2,3-trihydroxybenzene, nitrophenol, and bromophenol are reported. The aromatic alcohols interact with the framework by hydrogen bonding to oxygen atoms that bridge zinc and phosphorus atoms in the inorganic portion of the network. In addition, three hydrogen-bonded networks that contain trimethylene-dipyridine and either hydroquinone or 1,2,3-trihydroxybenzene are reported. In these structures, the aromatic alcohol acts as a hydrogen bond donor to trimethylene-dipyridine. The flexibility of trimethylene-dipyridine is important to the construction of the new frameworks. Calculations using PM5 parameters demonstrate that trimethylene-dipyridine can adopt a wide variety of conformations with a minimal energy expense.     

Phosphite uptake and distribution in potato tubers following foliar and postharvest applications of phosphite‐based fungicides for late blight control

Foliar and postharvest applications of phosphite (Phi)‐based fungicides are used to control the oomycete Phytophthora infestans which is responsible for the occurrence of late blight in potatoes. Optimisation of the usage of Phi‐based fungicides for disease control during the growing season and in subsequent storage can lead to improved potato production and processing quality. In order to assess the efficiency of Phi translocation to tubers, following foliar and postharvest treatments of potato crops with the Phi‐based fungicides, the amount of Phi in tubers was determined by a high‐performance ion chromatography method. The quantity of Phi found in tubers increased with the total amount of Phi‐based fungicides applied during the growing season. Foliar applications of Phi resulted in an uneven distribution of Phi in the three tuber regions analysed, with high concentrations being identified in the tuber cortex (32.5–166.4 µg g^?1 fresh tissue) and medulla regions followed by the skin area. Postharvest treatment of tubers led to a different distribution of Phi, with the highest concentrations of Phi found in the skin (411.0–876.6 µg g^?1 fresh tissue) followed by the cortex and medulla regions. As foliar treatments are essential to protect the aerial parts of the plants during the growth season, the best disease management practices of tubers should include the postharvest treatment in addition to foliar applications. The use of both types of treatments ensures that concentrations of Phi in excess of 100 µg g^?1 fresh tissue are present in tuber skin and cortex areas; such concentrations are needed to suppress the growth of P. infestans on tubers during storage.