Effects and mechanisms of glyphosate as phosphorus nutrient on element stoichiometry and metabolism in the diatom Phaeodactylum tricornutum.

The ability to utilize dissolved organic phosphorus (DOP) gives phytoplankton competitive advantages in P-limited environments. Our previous research indicates that the diatom Phaeodactylum tricornutum could grow on glyphosate, a DOP with carbon-phosphorus (C-P) bond and an herbicide, as sole P source. However, direct evidence and mechanism of glyphosate utilization are still lacking. In this study, using physiological and isotopic analysis, combined with transcriptomic profiling, we demonstrated the uptake of glyphosate by P. tricornutum and revealed the candidate responsible genes. Our data showed a low efficiency of glyphosate utilization by P. tricornutum, suggesting that glyphosate utilization costs energy and that the alga possessed an herbicide-resistant type of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. Compared to the P-limited cultures, the glyphosate-grown P. tricornutum cells up-regulated genes involved in DNA replication, cell growth, transcription, translation, carbon metabolism, and many genes encoding antioxidants. Additionally, cellular C and silicon (Si) increased remarkably while cellular nitrogen (N) declined in the glyphosate-grown P. tricornutum, leading to higher Si:C and Si:N ratios, which corresponded to the up-regulation of genes involved in the C metabolism and Si uptake and the down-regulation of those encoding N uptake. This has the potential to enhance C and Si export to the deep sea when P is limited but phosphonate is available. In sum, our study documented how P. tricornutum could utilize the herbicide glyphosate as P nutrient and how glyphosate utilization may affect the element content and stoichiometry in this diatom, which have important ecological implications in the future ocean.IMPORTANCEGlyphosate is the most widely used herbicide in the world and could be utilized as phosphorus (P) source by some bacteria. Our study first revealed that glyphosate could be transported into Phaeodactylum tricornutum cells for utilization and identified putative genes responsible for glyphosate uptake. This uncovers an alternative strategy of phytoplankton to cope with P deficiency considering phosphonate accounts for about 25% of the total dissolved organic phosphorus (DOP) in the ocean. Additionally, accumulation of carbon (C) and silicon (Si), as well as elevation of Si:C ratio in P. tricornutum cells when grown on glyphosate indicates glyphosate as the source of P nutrient has the potential to result in more C and Si export into the deep ocean. This, along with the differential ability to utilize glyphosate among different species, glyphosate supply in dissolved inorganic phosphorus (DIP)-depleted ecosystems may cause changes in phytoplankton community structure. These insights have implications in evaluating the effects of human activities (use of Roundup) and climate change (potentially reducing DIP supply in sunlit layer) on phytoplankton in the future ocean.

Biodegradation of selected aminophosphonates by the bacterial isolate Ochrobactrum sp. BTU1.

Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding. This study illustrates that widely used and structurally more complex aminophosphonates can be degraded by Ochrobactrum sp. BTU1 via the well-known degradation pathways but with different initial cleavage strategy compared to GS.

Novel standard biodegradation test for synthetic phosphonates.

Determination of biodegradation of synthetic phosphonates such as aminotris(methylenephosphonic acid) (ATMP), ethylenediamine tetra(methylenephosphonic acid) (EDTMP), or diethylenetriamine penta(methylenephosphonic acid) (DTPMP) is a great challenge. Commonly, ready biodegradability of organic substances is assessed by OECD 301 standard tests. However, due to the chemical imbalance of carbon to phosphorus synthetic phosphonates do not promote microbial growth and, thus, limiting its biodegradation. Therefore, standard OECD test methods are not always reliable to predict the real biodegradability of phosphonates. In the presented study, we report the development of a standardized batch system suitable to synthetic phosphonates such as ATMP, EDTMP, DTPMP and others. The novel standard batch test is applicable with pure strains, activated sludge from different wastewater treatment plants (i.e., municipal and industrial), and with tap water as inoculum. We optimized the required calcium and magnesium exposure levels as well as the amount of the start inoculum biomass. We demonstrated that our test also allows to determine several parameters including ortho-phosphate (o-PO43-), total phosphorus (TP), ammonium (NH4+) and total organic carbon (TOC). In addition, also LC/MS analyses of cell-free medium is applicable for determining the mother compounds and metabolites. We applied our optimized standardized batch with selected phosphonates and evidenced that the chemical structure has a major influence of the microbial growth rates. Thus, our novel batch test overcomes drawbacks of the OECD 301 test series for determination of easy biodegradability for stoichiometric imbalanced organic compounds such as phosphonates.

The facilitating role of phycospheric heterotrophic bacteria in cyanobacterial phosphonate availability and Microcystis bloom maintenance.

BACKGROUND: Phosphonates are the main components in the global phosphorus redox cycle. Little is known about phosphonate metabolism in freshwater ecosystems, although rapid consumption of phosphonates has been observed frequently. Cyanobacteria are often the dominant primary producers in freshwaters; yet, only a few strains of cyanobacteria encode phosphonate-degrading (C-P lyase) gene clusters. The phycosphere is defined as the microenvironment in which extensive phytoplankton and heterotrophic bacteria interactions occur. It has been demonstrated that phytoplankton may recruit phycospheric bacteria based on their own needs. Therefore, the establishment of a phycospheric community rich in phosphonate-degrading-bacteria likely facilitates cyanobacterial proliferation, especially in waters with scarce phosphorus. We characterized the distribution of heterotrophic phosphonate-degrading bacteria in field Microcystis bloom samples and in laboratory cyanobacteria "phycospheres" by qPCR and metagenomic analyses. The role of phosphonate-degrading phycospheric bacteria in cyanobacterial proliferation was determined through coculturing of heterotrophic bacteria with an axenic Microcystis aeruginosa strain and by metatranscriptomic analysis using field Microcystis aggregate samples. RESULTS: Abundant bacteria that carry C-P lyase clusters were identified in plankton samples from freshwater Lakes Dianchi and Taihu during Microcystis bloom periods. Metagenomic analysis of 162 non-axenic laboratory strains of cyanobacteria (consortia cultures containing heterotrophic bacteria) showed that 20% (128/647) of high-quality bins from eighty of these consortia encode intact C-P lyase clusters, with an abundance ranging up to nearly 13%. Phycospheric bacterial phosphonate catabolism genes were expressed continually across bloom seasons, as demonstrated through metatranscriptomic analysis using sixteen field Microcystis aggregate samples. Coculturing experiments revealed that although Microcystis cultures did not catabolize methylphosphonate when axenic, they demonstrated sustained growth when cocultured with phosphonate-utilizing phycospheric bacteria in medium containing methylphosphonate as the sole source of phosphorus. CONCLUSIONS: The recruitment of heterotrophic phosphonate-degrading phycospheric bacteria by cyanobacteria is a hedge against phosphorus scarcity by facilitating phosphonate availability. Cyanobacterial consortia are likely primary contributors to aquatic phosphonate mineralization, thereby facilitating sustained cyanobacterial growth, and even bloom maintenance, in phosphate-deficient waters. Video Abstract.

The functional importance of bacterial oxidative phosphonate pathways.

Organophosphonates (Pns) are a unique class of natural products characterized by a highly stable C-P bond. Pns exhibit a wide array of interesting structures as well as useful bioactivities ranging from antibacterial to herbicidal. More structurally simple Pns are scavenged and catabolized by bacteria as a source of phosphorus. Despite their environmental and industrial importance, the pathways involved in the metabolism of Pns are far from being fully elucidated. Pathways that have been characterized often reveal unusual chemical transformations and new enzyme mechanisms. Among these, oxidative enzymes play an outstanding role during the biosynthesis and degradation of Pns. They are to a high extent responsible for the structural diversity of Pn secondary metabolites and for the break-down of both man-made and biogenic Pns. Here, we review our current understanding of the importance of oxidative enzymes for microbial Pn metabolism, discuss the underlying mechanistic principles, similarities, and differences between pathways. This review illustrates Pn biochemistry to involve a mix of classical redox biochemistry and unique oxidative reactions, including ring formations, rearrangements, and desaturations. Many of these reactions are mediated by specialized iron-dependent oxygenases and oxidases. Such enzymes are the key to both early pathway diversification and late-stage functionalization of complex Pns.

Transcriptomic-Guided Phosphonate Utilization Analysis Unveils Evidence of Clathrin-Mediated Endocytosis and Phospholipid Synthesis in the Model Diatom, Phaeodactylum tricornutum.

Phosphonates are important components of marine organic phosphorus, but their bioavailability and catabolism by eukaryotic phytoplankton remain enigmatic. Here, diatom Phaeodactylum tricornutum was used to investigate the bioavailability of phosphonates and describe the underlying molecular mechanism. The results showed that 2-aminoethylphosphonic acid (2-AEP) can be utilized as an alternative phosphorus source. Comparative transcriptomics revealed that the utilization of 2-AEP comprised 2 steps, including molecular uptake through clathrin-mediated endocytosis and incorporation into the membrane phospholipids in the form of diacylglyceryl-2-AEP (DAG-2-AEP). In the global ocean, we found the prevalence and dynamic expression pattern of key genes that are responsible for vesicle formation (CLTC, AP-2) and DAG-AEP synthesis (PCYT2, EPT1) in diatom assemblages. This study elucidates a distinctive mechanism of phosphonate utilization by diatoms, and discusses the ecological implications. IMPORTANCE Phosphonates contribute ~25% of total dissolved organic phosphorus in the ocean, and are found to be important for marine phosphorus biogeochemical cycle. As a type of biogenic phosphonate produced by microorganisms, 2-aminoethylphosphonic acid (2-AEP) widely exists in the ocean. It is well known that 2-AEP can be cleaved and utilized by prokaryotes, but its ability to support the growth of eukaryotic phytoplankton remains unclear. Our research identified the bioavailability of 2-AEP for the diatom Phaeodactylum tricornutum, and proposed a distinctive metabolic pathway of 2-AEP utilization. Different from the enzymatic hydrolysis of phosphonates, the results suggested that P. tricornutum utilizes 2-AEP by incorporating it into phospholipid instead of cleaving the C-P bond. Moreover, the ubiquitous distribution of associated representative gene transcripts in the environmental assemblages and the higher gene transcript abundance in the cold regions were observed, which suggests the possible environmental adaption of 2-AEP utilization by diatoms.

Sustained stoichiometric imbalance and its ecological consequences in a large oligotrophic lake.

Considerable attention is given to absolute nutrient levels in lakes, rivers, and oceans, but less is paid to their relative concentrations, their nitrogen:phosphorus (N:P) stoichiometry, and the consequences of imbalanced stoichiometry. Here, we report 38 y of nutrient dynamics in Flathead Lake, a large oligotrophic lake in Montana, and its inflows. While nutrient levels were low, the lake had sustained high total N: total P ratios (TN:TP: 60 to 90:1 molar) throughout the observation period. N and P loading to the lake as well as loading N:P ratios varied considerably among years but showed no systematic long-term trend. Surprisingly, TN:TP ratios in river inflows were consistently lower than in the lake, suggesting that forms of P in riverine loading are removed preferentially to N. In-lake processes, such as differential sedimentation of P relative to N or accumulation of fixed N in excess of denitrification, likely also operate to maintain the lake's high TN:TP ratios. Regardless of causes, the lake's stoichiometric imbalance is manifested in P limitation of phytoplankton growth during early and midsummer, resulting in high C:P and N:P ratios in suspended particulate matter that propagate P limitation to zooplankton. Finally, the lake's imbalanced N:P stoichiometry appears to raise the potential for aerobic methane production via metabolism of phosphonate compounds by P-limited microbes. These data highlight the importance of not only absolute N and P levels in aquatic ecosystems, but also their stoichiometric balance, and they call attention to potential management implications of high N:P ratios.

2-Aminoethylphosphonate utilization in Pseudomonas putida BIRD-1 is controlled by multiple master regulators.

Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism.

A Phosphonate Natural Product Made by Pantoea ananatis is Necessary and Sufficient for the Hallmark Lesions of Onion Center Rot.

Pantoea ananatis is the primary cause of onion center rot. Genetic data suggest that a phosphonic acid natural product is required for pathogenesis; however, the nature of the molecule is unknown. Here, we show that P. ananatis produces at least three phosphonates, two of which were purified and structurally characterized. The first, designated pantaphos, was shown to be 2-(hydroxy[phosphono]methyl)maleate; the second, a probable biosynthetic precursor, was shown to be 2-(phosphonomethyl)maleate. Purified pantaphos is both necessary and sufficient for the hallmark lesions of onion center rot. Moreover, when tested against mustard seedlings, the phytotoxic activity of pantaphos was comparable to the widely used herbicides glyphosate and phosphinothricin. Pantaphos was also active against a variety of human cell lines but was significantly more toxic to glioblastoma cells. Pantaphos showed little activity when tested against a variety of bacteria and fungi.IMPORTANCEPantoea ananatis is a significant plant pathogen that targets a number of important crops, a problem that is compounded by the absence of effective treatments to prevent its spread. Our identification of pantaphos as the key virulence factor in onion center rot suggests a variety of approaches that could be employed to address this significant plant disease. Moreover, the general phytotoxicity of the molecule suggests that it could be developed into an effective herbicide to counter the alarming rise in herbicide-resistant weeds.

Phosphate-limited ocean regions select for bacterial populations enriched in the carbon-phosphorus lyase pathway for phosphonate degradation.

In tropical and subtropical oceanic surface waters phosphate scarcity can limit microbial productivity. However, these environments also have bioavailable forms of phosphorus incorporated into dissolved organic matter (DOM) that microbes with the necessary transport and hydrolysis metabolic pathways can access to supplement their phosphorus requirements. In this study we evaluated how the environment shapes the abundance and taxonomic distribution of the bacterial carbon-phosphorus (C-P) lyase pathway, an enzyme complex evolved to extract phosphate from phosphonates. Phosphonates are organophosphorus compounds characterized by a highly stable C-P bond and are enriched in marine DOM. Similar to other known bacterial adaptions to low phosphate environments, C-P lyase was found to become more prevalent as phosphate concentrations decreased. C-P lyase was particularly enriched in the Mediterranean Sea and North Atlantic Ocean, two regions that feature sustained periods of phosphate depletion. In these regions, C-P lyase was prevalent in several lineages of Alphaproteobacteria (Pelagibacter, SAR116, Roseobacter and Rhodospirillales), Gammaproteobacteria, and Actinobacteria. The global scope of this analysis supports previous studies that infer phosphonate catabolism via C-P lyase is an important adaptive strategy implemented by bacteria to alleviate phosphate limitation and expands the known geographic extent and taxonomic affiliation of this metabolic pathway in the ocean.

N-phosphonomethylglycine utilization by the psychrotolerant yeast Solicoccozyma terricola M 3.1.4.

Solicoccozyma terricola M 3.1.4., the yeast strain isolated from soil sample from blueberry cultivation in Miedzyrzec Podlaski in Poland, is capable to split of phosphorus to nitrogen and nitrogen to carbon bonds in N-phosphonomethylglycine (PMG, glyphosate). The biodegradation process proceeds in the phosphate-independent manner. It is the first example of a psychrotolerant yeast strain able to degrade PMG via CN bond cleavage accompanied by AMPA formation and not like in most microorganisms via CP bond disruption followed by the sarcosine pathway. Glyphosate oxidoreductase (GOX) type activity was detected in cell-free extracts prepared from S. terricola M 3.1.4. pregrown on 4 mM PMG as a sole phosphorus and nitrogen source in cultivation medium.

The Abc of Phosphonate Breakdown: A Mechanism for Bacterial Survival.

Bacteria have evolved advanced strategies for surviving during nutritional stress, including expression of specialized enzyme systems that allow them to grow on unusual nutrient sources. Inorganic phosphate (Pi ) is limiting in most ecosystems, hence organisms have developed a sophisticated, enzymatic machinery known as carbon-phosphorus (C-P) lyase, allowing them to extract phosphate from a wide range of phosphonate compounds. These are characterized by a stable covalent bond between carbon and phosphorus making them very hard to break down. Despite the challenges involved in both synthesizing and catabolizing phosphonates, they are widespread in nature. The enzymes required for the bacterial C-P lyase pathway have been identified and for the most part structurally characterized. Nevertheless, the mechanistic principles governing breakdown of phosphonate compounds remain enigmatic. In this review, an overview of the C-P lyase pathway is provided and structural aspects of the involved enzyme complexes are discussed with a special emphasis on the role of ATP-binding cassette (ABC) proteins.

Center Rot of Onion (Allium cepa) Caused by Pantoea ananatis Requires pepM, a Predicted Phosphonate-Related Gene.

Pantoea ananatis, a cause of center rot of onion, is problematic in the United States and elsewhere. The bacterium lacks disease determinants common to most other bacterial pathogens of plants. A genomic island containing the gene pepM was detected within many onion-pathogenic strains of P. ananatis of diverse origins. The pepM gene of P. ananatis putatively encodes a protein that converts phosphoenolpyruvate to phosphonopyruvate, the first step in the biosynthesis of phosphonates and related molecules. This gene appears to be essential for center rot disease. Deletion of pepM rendered the mutant strain unable to cause lesions in leaves of growing onions and water-soaking of inoculated yellow onion bulbs. Furthermore, growth of the deletion mutant in onion leaves was significantly diminished compared with wild-type bacteria, and the mutant failed to cause cell death in tobacco. Complementation of the mutated strain with pepM restored the phenotype to wild-type capability. The pepM gene is the first pathogenicity factor identified that affects bacterial fitness as well as symptom development in both leaves and bulbs in a pathogen causing center rot of onion.

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 µm pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 °C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

Synthetic and Natural Lipase Inhibitors.

Lipases are enzymes that catalyse the hydrolysis of ester bonds of triglycerides ranging among biocatalysts of considerable physiological significance and industrial potential. Better understanding of the catalytic functions and achieving the possibility to control the biocatalysis process, in particular exploring some activators and inhibitors of lipases, seems to be crucial in the context of novel applications. The lipase activity is a function of interfacial composition: the enzyme can be there activated as well as denaturated or deactivated and the interface is an appropriate site for modulating lipolysis. Lipase inhibitor, interacts directly with the enzyme and inhibits lipase action. Alternatively, some compounds can postpone the lipolytic reaction via adsorption to the interphase or to the substrate molecules. The aim of this review is to summarise the current knowledge concerning human, animal and microbial lipase inhibitors, which were grouped into two categories: synthetic lipase inhibitors (including phosphonates, boronic acids and fats analogues) and natural compounds (including ß-lactones and some botanical foodstuffs - plant extracts and plant metabolites, mainly polyphenols and saponins as well as peptides and some dietary fibers). The topics discussed include also inhibition issues from the viewpoint of obesity treatment. Among natural compounds able to inhibit lipase activity are ß- lactones including orlistat. Orlistat is the only registered drug for obesity treatment in many countries and lipases are essential enzymes for lipid absorption - thus fat absorption or obesity can be controlled by lipase inhibition, especially pancreatic lipase which is responsible for the hydrolysis of over 80% of total dietary fats. Its effectiveness in obesity treatment was also described.

On the use of 31P NMR for the quantification of hydrosoluble phosphorus-containing compounds in coral host tissues and cultured zooxanthellae.

(31)P Nuclear Magnetic Resonance (NMR) was assessed to investigate the phosphorus-containing compounds present in the tissues of the scleractinian coral Stylophora pistillata as well as of cultured zooxanthellae (CZ). Results showed that phosphorus-containing compounds observed in CZ were mainly phosphate and phosphate esters. Phosphate accounted for 19 ± 2% of the total phosphorus compounds observed in CZ maintained under low P-levels (0.02 µM). Adding 5 mM of dissolved inorganic phosphorus (KH2PO4) to the CZ culture medium led to a 3.1-fold increase in intracellular phosphate, while adding 5 mM of dissolved organic phosphorus led to a reduction in the concentration of phosphorus compounds, including a 2.5-fold intracellular phosphate decrease. In sharp contrast to zooxanthellae, the host mainly contained phosphonates, and to a lesser extent, phosphate esters and phosphate. Two-months of host starvation decreased the phosphate content by 2.4 fold, while bleaching of fed corals did not modify this content. Based on (31)P NMR analyses, this study highlights the importance of phosphonates in the composition of coral host tissues, and illustrates the impact of phosphorus availability on the phosphorus composition of host tissues and CZ, both through feeding of the host and inorganic phosphorus enrichment of the CZ.

Structural insights into the bacterial carbon-phosphorus lyase machinery.

Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.

The overproduction of 2,4-DTBP accompanying to the lack of available form of phosphorus during the biodegradative utilization of aminophosphonates by Aspergillus terreus.

Although information about the ability of some filamentous fungi to biodegrade organophosphonates is available, the knowledge about accompanying changes in fungal metabolism is very limited. The aim of our study was to determine the utilization of the chosen, structurally diverse aminophosphonates by Aspergillus terreus (Thom), in the context of the behaviour of this fungus while growing in unfavourable conditions, namely the lack of easily available phosphates. We found that all the studied compounds were utilized by fungus as nutritive sources of phosphorus, however, their effect on the production of fungal biomass depended on their structure. We also observed an interesting change in the metabolism of A. terreus; namely the overproduction of 2,4-di-tert-butylphenol (2,4-DTBP), which is known to possess fungistatic activity. In the case of our study, the biosynthesis of this compound was induced by phosphorus starvation, caused either by the lack of that element in the medium, or the poor degradation of phosphonate.

Organophosphonates revealed: new insights into the microbial metabolism of ancient molecules.

Organophosphonates are ancient molecules that contain the chemically stable C-P bond, which is considered a relic of the reducing atmosphere on primitive earth. Synthetic phosphonates now have a wide range of applications in the agricultural, chemical and pharmaceutical industries. However, the existence of C-P compounds as contemporary biogenic molecules was not discovered until 1959, with the identification of 2-aminoethylphosphonic acid in rumen protozoa. Here, we review advances in our understanding of the biochemistry and genetics of microbial phosphonate metabolism, and discuss the role of these compounds and of the organisms engaged in their turnover within the P cycle.

Sublethal detergent concentrations increase metabolization of recalcitrant polyphosphonates by the cyanobacterium Spirulina platensis.

As a consequence of increasing industrial applications, thousand tons of polyphosphonates are introduced every year into the environment. The inherent stability of the C-P bond results in a prolonged half-life. Moreover, low uptake rates limit further their microbial metabolization. To assess whether low detergent concentrations were able to increase polyphosphonate utilization by the cyanobacterium Spirulina platensis, tolerance limits to the exposure to various detergents were determined by measuring the growth rate in the presence of graded levels below the critical micellar concentration. Then, the amount of hexamethylenediamine-N,N,N',N'-tetrakis(methylphosphonic acid) that is metabolized in the absence or in the presence of sublethal detergent concentrations was quantified by (31)P NMR analysis on either P-starved or P-fed cyanobacterial cultures. The strain tolerated the presence of detergents in the order: nonionic > anionic > cationic. When added to the culture medium at the highest concentrations showing no detrimental effects upon cell viability, detergents either improved or decreased polyphosphonate utilization, the anionic sodium dodecyl sulfate being the most beneficial. Metabolization was not lower in P-fed cells--a result that strengthens the possibility of using, in the future, this strain for bioremediation purposes.