Bioactive Phosphorus Dendrimers as a Universal Protein Delivery System for Enhanced Anti-inflammation Therapy.

Nanocarrier-based cytoplasmic protein delivery offers opportunities to develop protein therapeutics; however, many delivery systems are positively charged, causing severe toxic effects. For enhanced therapeutics, it is also of great importance to design nanocarriers with intrinsic bioactivity that can be integrated with protein drugs due to the limited bioactivity of proteins alone for disease treatment. We report here a protein delivery system based on anionic phosphite-terminated phosphorus dendrimers with intrinsic anti-inflammatory activity. A phosphorus dendrimer termed AK-137 with optimized anti-inflammatory activity was selected to complex proteins through various physical interactions. Model proteins such as bovine serum albumin, ribonuclease A, ovalbumin, and fibronectin (FN) can be transfected into cells to exert their respective functions, including cancer cell apoptosis, dendritic cell maturation, or macrophage immunomodulation. Particularly, the constructed AK-137@FN nanocomplexes display powerful therapeutic effects in acute lung injury and acute gout arthritis models by integrating the anti-inflammatory activity of both the carrier and protein. The developed anionic phosphite-terminated phosphorus dendrimers may be employed as a universal carrier for protein delivery and particularly utilized to deliver proteins and fight different inflammatory diseases with enhanced therapeutic efficacy.

Anaerobic dissimilatory phosphite oxidation, an extremely efficient concept of microbial electron economy.

Phosphite is a stable phosphorus compound that, together with phosphate, made up a substantial part of the total phosphorus content of the prebiotic Earth's crust. Oxidation of phosphite to phosphate releases electrons at an unusually low redox potential (-690 mV at pH 7.0). Numerous aerobic and anaerobic bacteria use phosphite as a phosphorus source and oxidise it to phosphate for synthesis of nucleotides and other phosphorus-containing cell constituents. Only two pure cultures of strictly anaerobic bacteria have been isolated so far that use phosphite as an electron donor in their energy metabolism, the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. The key enzyme of this metabolism is an NAD+ -dependent phosphite dehydrogenase enzyme that phosphorylates AMP to ADP. These phosphorylating phosphite dehydrogenases were found to be related to nucleoside diphosphate sugar epimerases. The produced NADH is channelled into autotrophic CO2 fixation via the Wood-Ljungdahl (CO-DH) pathway, thus allowing for nearly complete assimilation of the substrate electrons into bacterial biomass. This extremely efficient type of electron flow connects energy and carbon metabolism directly through NADH and might have been important in the early evolution of life when phosphite was easily available on Earth.

A fluorometric assay for high-throughput phosphite quantitation in biological and environmental matrices.

Phosphite, the anion of phosphorus acid, is an important metabolite in the global biogeochemical phosphorus cycle and a phosphorus species with unique agricultural properties. As such, methods for detecting phosphite quantitatively and selectively are critical to evidencing phosphorus redox chemistry. Here, we present a fluorescence-based assay for phosphite, utilizing the NAD+-dependent oxidation of phosphite by phosphite dehydrogenase and the subsequent reduction of resazurin to resorufin. With the application of a thermostable phosphite dehydrogenase, a medium-invariant analytical approach, and novel sample preparation methods, the assay is capable of rapid and accurate phosphite quantification with a 3 µM limit of detection in a wide array of biologically- and environmentally-relevant matrices, including bacterial and archaeal cell lysate, seawater, anaerobic digester sludge, and plant tissue. We demonstrate the utility of the assay by quantitating phosphite uptake in a model crop plant in the presence or absence of a phosphite-oxidising strain of Pseudomonas stutzeri as a soil additive, establishing this bacterium as an efficient phosphite converting biofertilizer.

Marine picocyanobacterial PhnD1 shows specificity for various phosphorus sources but likely represents a constitutive inorganic phosphate transporter.

Despite being fundamental to multiple biological processes, phosphorus (P) availability in marine environments is often growth-limiting, with generally low surface concentrations. Picocyanobacteria strains encode a putative ABC-type phosphite/phosphate/phosphonate transporter, phnDCE, thought to provide access to an alternative phosphorus pool. This, however, is paradoxical given most picocyanobacterial strains lack known phosphite degradation or carbon-phosphate lyase pathway to utilise alternate phosphorus pools. To understand the function of the PhnDCE transport system and its ecological consequences, we characterised the PhnD1 binding proteins from four distinct marine Synechococcus isolates (CC9311, CC9605, MITS9220, and WH8102). We show the Synechococcus PhnD1 proteins selectively bind phosphorus compounds with a stronger affinity for phosphite than for phosphate or methyl phosphonate. However, based on our comprehensive ligand screening and growth experiments showing Synechococcus strains WH8102 and MITS9220 cannot utilise phosphite or methylphosphonate as a sole phosphorus source, we hypothesise that the picocyanobacterial PhnDCE transporter is a constitutively expressed, medium-affinity phosphate transporter, and the measured affinity of PhnD1 to phosphite or methyl phosphonate is fortuitous. Our MITS9220_PhnD1 structure explains the comparatively lower affinity of picocyanobacterial PhnD1 for phosphate, resulting from a more limited H-bond network. We propose two possible physiological roles for PhnD1. First, it could function in phospholipid recycling, working together with the predicted phospholipase, TesA, and alkaline phosphatase. Second, by having multiple transporters for P (PhnDCE and Pst), picocyanobacteria could balance the need for rapid transport during transient episodes of higher P availability in the environment, with the need for efficient P utilisation in typical phosphate-deplete conditions.

Potassium Phosphite Activates Components Associated with Constitutive Defense Responses in Coffea arabica Cultivars.

Phosphites have been used as inducers of resistance, activating the defense of plants and increasing its ability to respond to the invasion of the pathogen. However, the mode of action of phosphites in defense responses has not yet been fully elucidated. The objective of this study was to evaluate the effect of potassium phosphite (KPhi) in coffee cultivars with different levels of resistance to rust to clarify the mechanism by which KPhi activates the constitutive defense of plants. To this end, we studied the expression of genes and the activity of enzymes involved in the defense pathway of salicylic acid (SA) and reactive oxygen species (ROS), in addition to the levels of total soluble phenolic compounds and soluble lignin. Treatment with KPhi induced constitutive defense responses in cultivars resistant and susceptible to rust. The results suggest that KPhi acts in two parallel defense pathways, SA and ROS, which are essential for the induction of systemic acquired resistance (SAR) when activated simultaneously. The activation of the mechanisms associated with defense routes demonstrates that KPhi is a potential inducer of resistance in coffee plants.

Gatekeeper Residue Replacement in a Phosphite Transporter Enhances Mutational Robustness of the Biocontainment Strategy.

Biocontainment is a key methodology to reduce environmental risk through the deliberate release of genetically modified microorganisms. Previously, we developed a phosphite (HPO32-)-dependent biocontainment strategy, by expressing a phosphite-specific transporter HtxBCDE and phosphite dehydrogenase in bacteria devoid of their indigenous phosphate (HPO42-) transporters. This strategy did not allow Escherichia coli to generate escape mutants (EMs) in growth media containing phosphate as a phosphorus source using an assay with a detection limit of 1.9 × 10-13. In this study, we found that the coexistence of a high dose of phosphate (>0.5 mM) with phosphite in the growth medium allows the phosphite-dependent E. coli strain to generate EMs at a frequency of approximately 5.4 × 10-10. In all EMs, the mutation was a single amino acid substitution of phenylalanine to cysteine or serine at position 210 of HtxC, the transmembrane domain protein of the phosphorus compound transporter HtxBCDE. Replacement of the HtxC F210 residue with the other 17 amino acids revealed that HtxC F210 is crucial in determining substrate specificity of HtxBCDE. Based on the finding of the role of HtxC F210 as a "gatekeeper" residue for this transporter, we demonstrate that the replacement of HtxC F210 with amino acids resulting from codons that require two simultaneous point mutations to generate phosphate permissive HtxC mutants can reduce the rate of EM generation to an undetectable level. These findings also provide novel insights into the functional classification of HtxBCDE as a noncanonical ATP-binding cassette transporter in which the transmembrane domain protein participates in substrate recognition.

Synthesis, Structural Determination, and Antioxidant Activities of Acyclic and Substituted Heterocyclic Phosphonates Linearly Linked 4-hydroxy-2(1H)-quinolinone.

The chemical reactivity of 3-[(E)-3-(dimethylamino)-2-propenoyl]-4-hydroxy-1-methy-2(1H)-quinolinone (1) towards some phosphorus reagents was studied. The enaminone 1 was cyclized into pyranoquinolinylphosphonate 2 via treatment with diethyl phosphite in basic medium. However, its reaction with triethoxy phosphonoacetate gave the substituted oxopyranylphosphonate 3. Using the same reaction conditions, both thioxopyridinylphosphonate 4 and oxopyranylphosphonate 5 were produced via a reaction of enaminone 1 with both diethyl 2-amino-2-thioxoethylphosphonate and diethyl vinylphosphonate, respectively, in low yields. In addition, the two novel oxopyridinylphosphonates 6 and 7 were obtained by treatment of enaminone 1 with a diethyl cyanomethylphosphonate reagent. Two oaxathiaphosphininyl derivatives, 8 and 9, were obtained by treatment of the enaminone 1 with O, O-diethyl dithiophosphoric acid under different reaction conditions. Diazaphosphininyl 11 and oxazaphosphininyl 12 derivatives were obtained in excellent yields using a P-phenylphosphonic diamide reagent under different reaction conditions. The treatment of the enaminone 1 with phosphorus pentasulfide produced the non-phosphorylated product thioxothiopyranoquinolinone 13. Finally, the enaminone was turned into oxathiaphosphininyl 14 using Lawesson's reagent. The possible reaction mechanisms of the formation of these products were discussed. The structures of newly isolated products were established by elemental analysis and spectral tools. The compounds were evaluated for their antioxidant activities.

Comparative Transcriptome Profiling Reveals Potential Candidate Genes, Transcription Factors, and Biosynthetic Pathways for Phosphite Response in Potato (Solanum tuberosum L.).

The study was conducted with C31 and C80 genotypes of the potato (Solanum tuberosum L.), which are tolerant and susceptible to phosphite (Phi, H2PO3), respectively. To decipher the molecular mechanisms underlying tolerance and susceptibility to Phi in the potato, RNA sequencing was used to study the global transcriptional patterns of the two genotypes. Media were prepared with 0.25 and 0.50 mM Phi, No-phosphorus (P), and 1.25 mM (phosphate, Pi as control). The values of fragments per kilobase of exon per million mapped fragments of the samples were also subjected to a principal component analysis, grouping the biological replicates of each sample. Using stringent criteria, a minimum of 819 differential (DEGs) were detected in both C80-Phi-0.25_vs_C80-Phi-0.50 (comprising 517 upregulated and 302 downregulated) and C80-Phi-0.50_vs_C80-Phi-0.25 (comprising 302 upregulated and 517 downregulated) and a maximum of 5214 DEGs in both C31-Con_vs_C31-Phi-0.25 (comprising 1947 upregulated and 3267 downregulated) and C31-Phi-0.25_vs_C31-Con (comprising 3267 upregulated and 1947 downregulated). DEGs related to the ribosome, plant hormone signal transduction, photosynthesis, and plant-pathogen interaction performed important functions under Phi stress, as shown by the Kyoto Encyclopedia of Genes and Genomes annotation. The expressions of transcription factors increased significantly in C31 compared with C80. For example, the expressions of Soltu.DM.01G047240, Soltu.DM.08G015900, Soltu.DM.06G012130, and Soltu.DM.08G012710 increased under P deficiency conditions (Phi-0.25, Phi-0.50, and No-P) relative to the control (P sufficiency) in C31. This study adds to the growing body of transcriptome data on Phi stress and provides important clues to the Phi tolerance response of the C31 genotype.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida.

The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.

Borate and phosphite treatments of potato plants (Solanum tuberosum L.) as a proof of concept to reinforce the cell wall structure and reduce starch digestibility.

Potatoes are one of the main sources of carbohydrates in human diet, however they have a high glycaemic index (GI). Hence, developing new agricultural and industrial strategies to produce low GI potatoes represents a health priority to prevent obesity and related diseases. In this work, we investigated whether treatments of potato plants with elicitors of plant defence responses can lead to a reduction of tuber starch availability and digestibility, through the induction of cell wall remodelling and stiffening. Treatments with phosphites (KPhi) and borate were performed, as they are known to activate plant defence responses that cause modifications in the architecture and composition of the plant cell wall. Data of suberin autofluorescence demonstrated that potato plants grown in a nutrition medium supplemented with KPhi and borate produced tubers with a thicker periderm, while pectin staining demonstrated that KPhi treatment induced a reinforcement of the wall of storage parenchyma cells. Both compounds elicited the production of H2O2, which is usually involved in cell-wall remodelling and stiffening reactions while only KPhi caused an increase of the total content of phenolic compounds. A two-phase digestion in vitro assay showed that treatment with KPhi determined a significant decrease of the starch hydrolysis rate in potato tubers. This work highlights the ability of cell wall architecture in modulating starch accessibility to digestive enzymes, paving the way for new agronomic practices to produce low GI index potatoes.

The use of phosphonates in agriculture. Chemical, biological properties and legislative issues.

The Phosphorus (III) derivatives, named Phosphonates, include congeners with properties as fungicides that are effective in controlling Oomycetes. Examples are organic compounds like Fosetyl-Al [Aluminium tris-(ethylphosphonate)] and salts formed with the anion of phosphonic acid [(OH)2HPO] and Potassium, Sodium and Ammonium cations. According to IUPAC, the correct nomenclature for these compounds is "phosphonates", but in common language and scientific literature they are often named "phosphites", creating ambiguity. The European legislation restricts the use of phosphonates, with the ban for application in organic agriculture. However, phosphonate residues were detected in some organic products due to their addition to fertilizers allowed in organic agriculture. The legitimacy of this addition is controversial, as it is not evident if phosphonates have also a nutritional role in addition to their fungicidal properties. The new European Directive EU 1009/2019 resolves the problem by banning the phosphonates addition to fertilizers and placing a limit of 0.5% by mass for unintentional addition. However, an official method is not available for phosphonates determination in fertilizers and approval by the European Committee for Standardization (CEN) is necessary in a short time. This review presents an overview about the chemical, biological, analytical and legislative aspects of phosphonates and aims at providing: clarity on the correct nomenclature to avoid misunderstandings; the evaluation of phosphonates properties with the absence of a nutritional role, justifying the ban on adding to fertilizers; a summary of analytical techniques that could be considered by CEN to complete the analytical background for the agricultural use of phosphonates.

Valorization of calcium phosphite waste as phosphorus fertilizer: Effects on green manure productivity and soil properties.

The potential to use calcium phosphite (Ca-Phi) as phosphorus (P) fertilizer may represent an effective recycling of P-containing by-products. A greenhouse experiment was conducted to investigate the effect of Ca-Phi (38 kg P ha-1) on soil properties and the growth parameters of four green manure species in clay and sandy soils using Ca-Phi, TSP (triple superphosphate) and control (no fertilization) as treatments. Eight weeks after sowing, we measured aboveground biomass yield, phosphite (Phi) concentration in plant biomass, different soil P pools as well as microbial biomass nutrients. Compared to control, the addition of Ca-Phi did not negatively affect green manure yield, except for lupine (Lupinus albus L.) in clay soil. The Phi concentration in plant biomass varied across species and soil type with a maximum concentration of about 400 mg Phi kg-1 for mustard (Brassica juncea L.) in clay soil. Compared to control, TSP and Ca-Phi fertilization had a similar effect on different P pools and microbial biomass nutrients (C, N and P) although the response was soil-type dependent. In the sandy soil, after Ca-Phi addition the amount of available P (PNHCO3) increased to the same extent as in the TSP treatment (i.e. around 6 mg P kg-1) suggesting that Ca-Phi was, at least partly, oxidized. In the clay soil with high P fixing capacity, Ca-Phi promoted higher PNaHCO3 than TSP likely due to different solubility of chemical P forms. Additional studies are however required to better understand soil microbial responses and to quantify the P agronomical efficiency for the following crop under Ca-Phi fertilization.

Effects of phosphite as a plant biostimulant on metabolism and stress response for better plant performance in Solanum tuberosum.

Food availability represents a major worldwide concern due to population growth, increased demand, and climate change. Therefore, it is imperative to identify compounds that can improve crop performance. Plant biostimulants have gained prominence because of their potentials to increase germination, productivity and quality of a wide range of horticultural and agronomic crops. Phosphite (Phi), an analog of orthophosphate, is an emerging biostimulant used in horticulture and agronomy. The aim of this study was to uncover the molecular mechanisms through which Phi acts as a biostimulant with potential effects of overall plant growth. Field and greenhouse experiments, using 4 potato cultivars, showed that following Phi applications, plant performance, including several physio-biochemical traits, crop productivity, and quality traits, were significantly improved. RNA sequencing of control and Phi-treated plants of cultivar Xingjia No. 2, at 0 h, 6 h, 24 h, 48 h, 72 h and 96 h after the Phi application for 24 h revealed extensive changes in the gene expression profiles. A total of 2856 differentially expressed genes were identified, suggesting that multiple pathways of primary and secondary metabolism, such as flavonoids biosynthesis, starch and sucrose metabolism, and phenylpropanoid biosynthesis, were strongly influenced by foliar applications of Phi. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses associated with defense responses revealed significant effects of Phi on a plethora of defense mechanisms. These results suggest that Phi acted as a biostimulant by priming the plants, that was, by triggering dynamic changes in gene expression and modulating metabolic fluxes in a way that allowed plants to perform better. Therefore, Phi usage has the potential to improve crop yield and health, alleviating the challenges posed by the need of feeding a growing world population, while minimizing the agricultural impact on human health and environment.

Phosphite Integrated in Late Blight Treatment Strategies in Starch Potato Does Not Cause Residues in the Starch Product.

Currently available fungicides against potato late blight are effective but there are concerns about the sustainability of frequent applications and the risks of fungicide resistance. Therefore, we investigated how potassium phosphite can be integrated into late blight control programs with reduced fungicides in field trials. Phosphite was somewhat less effective than the conventional fungicides at suppressing late blight in the foliage, and the tubers contained less starch. However, when we reduced the amount of phosphite and combined it with reduced amounts of conventional fungicides, we observed no differences in disease suppression, total yields, and tuber starch contents compared with the full treatments with conventional fungicides. The amount of phosphite detected in the harvested tubers was linearly associated with the amount of phosphite applied to the foliage. Our analyses indicate that phosphite could replace some fungicides without exceeding the current European Union standards for the maximum residue levels in potato tubers. No phosphite was detected in the starch from the tubers. In 1 of 2 years, early blight (caused by Alternaria solani) was less severe in the phosphite treatments than in the treatments without phosphite. The integration of phosphite into current treatment strategies would reduce the dependence on conventional fungicides.

Redox mechanism and stability of uranyl phosphites at mineral surfaces: Cooperative proton/electron transfer and high efficacy for Uranium(VI) reduction.

Uranium phosphites have recently emerged as promising materials to remediate radioactive contamination. In this study, the redox mechanisms of uranyl phosphites at mineral surfaces have been addressed by periodic DFT calculations with dispersion corrections. Different from other ligands, the phosphite anions (H2PO3-, HPO32-) are efficient reducing agents for uranyl reduction, and the redox reactions are divided into three steps, as isomerization between two phosphite anion isomers (Step 1), conformational transition (Step 2) and dissociation of the water molecule (Step 3). A second water molecule is critical to lower the activation barriers of Step 1, and all activation barriers are moderate so that the redox reactions occur favorably under normal conditions, which are further dramatically accelerated by the highly exergonic Step 3. Accordingly, formation of uranyl phosphites becomes an effective approach to manage uranium pollution. Moreover, the lower activation barriers for H2PO3- rather than HPO32- rationalize the superior reduction activities of uranyl phosphites and the enhanced stability of U(IV) products at lower pH conditions. Owing to the cooperative proton/electron transfer, the U(VI) reduction to U(IV) and P(III) oxidation to P(V) are completed within one step, with transition states being featured by the U(V) and P(IV) species.

Exogenous phosphite application alleviates the adverse effects of heat stress and improves thermotolerance of potato (Solanum tuberosum L.) seedlings.

Phosphite (Phi), an analog of phosphate (Pi) anion, is emerging as a potential biostimulator, fungicide and insecticide. Here, we reported that Phi also significantly enhanced thermotolerance in potatoes under heat stress. Potato plants with and without Phi pretreatment were exposed to heat stress and their heat tolerance was examined by assessing the morphological characteristics, photosynthetic pigment content, photosystem II (PS II) efficiency, levels of oxidative stress, and level of DNA damage. In addition, RNA-sequencing (RNA-Seq) was adopted to investigate the roles of Phi signals and the underlying heat resistance mechanism. RNA-Seq revealed that Phi orchestrated plant immune responses against heat stress by reprograming global gene expressions. Results from physiological data combined with RNA-Seq suggested that the supply of Phi not only was essential for the better plant performance, but also improved thermotolerance of the plants by alleviating oxidative stress and DNA damage, and improved biosynthesis of osmolytes and defense metabolites when exposed to unfavorable thermal conditions. This is the first study to explore the role of Phi in thermotolerance in plants, and the work can be applied to other crops under the challenging environment.

Identification of potassium phosphite responsive miRNAs and their targets in potato.

Micro RNAs (miRNAs) are small single strand non-coding RNAs that regulate gene expression at the post-transcriptional level, either by translational inhibition or mRNA degradation based on the extent of complementarity between the miRNA and its target mRNAs. Potato (Solanum tuberosum L.) is the most important horticultural crop in Argentina. Achieving an integrated control of diseases is crucial for this crop, where frequent agrochemical applications, particularly fungicides, are carried out. A promising strategy is based on promoting induced resistance through the application of environmentally friendly compounds such as phosphites, inorganic salts of phosphorous acid. The use of phosphites in disease control management has proven to be effective. Although the mechanisms underlying their effect remain unclear, we postulated that miRNAs could be involved. Therefore we performed next generation sequencing (NGS) in potato leaves treated and non treated with potassium phosphite (KPhi). We identified 25 miRNAs that were expressed differentially, 14 already annotated in miRBase and 11 mapped to the potato genome as potential new miRNAs. A prediction of miRNA targets showed genes related to pathogen resistance, transcription factors, and oxidative stress. We also analyzed in silico stress and phytohormone responsive cis-acting elements on differentially expressed pre miRNAs. Despite the fact that some of the differentially expressed miRNAs have been already identified, this is to our knowledge the first report identifying miRNAs responsive to a biocompatible stress resistance inducer such as potassium phosphite, in plants. Further characterization of these miRNAs and their target genes might help to elucidate the molecular mechanisms underlying KPhi-induced resistance.

Phosphite binding by the HtxB periplasmic binding protein depends on the protonation state of the ligand.

Phosphorus acquisition is critical for life. In low phosphate conditions, some species of bacteria have evolved mechanisms to import reduced phosphorus compounds, such as phosphite and hypophosphite, as alternative phosphorus sources. Uptake is facilitated by high-affinity periplasmic binding proteins (PBPs) that bind cargo in the periplasm and shuttle it to an ATP-binding cassette (ABC)-transporter in the bacterial inner membrane. PtxB and HtxB are the PBPs responsible for binding phosphite and hypophosphite, respectively. They recognize the P-H bond of phosphite/hypophosphite via a conserved P-H...π interaction, which confers nanomolar dissociation constants for their respective ligands. PtxB also has a low-level binding affinity for phosphate and hypophosphite, whilst HtxB can facilitate phosphite uptake in vivo. However, HtxB does not bind phosphate, thus the HtxBCDE transporter has recently been successfully exploited for biocontainment of genetically modified organisms by phosphite-dependent growth. Here we use a combination of X-ray crystallography, NMR and Microscale Thermophoresis to show that phosphite binding to HtxB depends on the protonation state of the ligand, suggesting that pH may effect the efficiency of phosphite uptake by HtxB in biotechnology applications.

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

[Establishment of a plant phosphorus utilization and weed control system based on phosphite and its dehydrogenase].

Nowadays, available phosphorus (P) deficiency in soil and weed resistance to herbicides have emerged as two severe limiting factors for sustainable agriculture. Therefore, it is of urgent needs to improve plant absorption/utilization ability of the soil P, seek phosphate (Pi)-alternative P fertilizers, and develop new forms of weed control systems. Phosphite (Phi), as a P resource of relatively high amount only less than Pi in Earth, can be converted to utilizable Pi uniquely in some bacterial species by oxidization via its specific dehydrogenase (PTDH), but inhibits plant growth and development. This implies that Phi might rather become a suitable P fertilizer for plants if introducing a PTDH detoxifier from bacteria. Herein, we created the transgenic tobaccos harboring a Pseudomonas PTDH gene (PsPtx) amplified from the soil metagenome previously. RT-PCR showed that the exotic PsPtx gene could express similarly in root, stem and leaf tissues of all transgenic lines. PsPtx transgenic tobaccos could utilize Phi by oxidization as the sole Pi supply, and also outperformed wild-type tobacco with a remarkably dominant growth under Phi stress conditions. Moreover, the PsPtx gene was preliminarily evaluated with a notable quality as a potential candidate of the selection marker in plant genetic transformation. Conclusively, PsPtx and its encoded phosphite dehydrogenase might be applicable for developing a dual system of plant phosphorus utilization and weed control using Phi as P fertilizer and herbicide, and provide an effectual solution to some obstacles in the current crop transgenic studies.