The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

Phosphorus uptake, partitioning and redistribution during grain filling in rice.

BACKGROUNDS AND AIMS: In cultivated rice, phosphorus (P) in grains originates from two possible sources, namely exogenous (post-flowering root P uptake from soil) or endogenous (P remobilization from vegetative parts) sources. This study investigates P partitioning and remobilization in rice plants throughout grain filling to resolve contributions of P sources to grain P levels in rice. METHODS: Rice plants (Oryza sativa 'IR64') were grown under P-sufficient or P-deficient conditions in the field and in hydroponics. Post-flowering uptake, partitioning and re-partitioning of P was investigated by quantifying tissue P levels over the grain filling period in the field conditions, and by employing 33P isotope as a tracer in the hydroponic study. KEY RESULTS: Post-flowering P uptake represented 40-70 % of the aerial plant P accumulation at maturity. The panicle was the main P sink in all studies, and the amount of P potentially remobilized from vegetative tissues to the panicle during grain filling was around 20 % of the total aerial P measured at flowering. In hydroponics, less than 20 % of the P tracer taken up at 9 d after flowering (DAF) was found in the above-ground tissues at 14 DAF and half of it was partitioned to the panicle in both P treatments. CONCLUSIONS: The results demonstrate that P uptake from the soil during grain filling is a critical contributor to the P content in grains in irrigated rice. The P tracer study suggests that the mechanism of P loading into grains involves little direct transfer of post-flowering P uptake to the grain but rather substantial mobilization of P that was previously taken up and stored in vegetative tissues.

Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1.

SAMHD1 is a host protein responsible, at least in part, for the inefficient infection of dendritic, myeloid, and resting T cells by HIV-1. Interestingly, HIV-2 and SIVsm viruses are able to counteract SAMHD1 by targeting it for proteasomal degradation using their Vpx proteins. It has been proposed that SAMHD1 is a dGTP-dependent deoxynucleoside triphosphohydrolase (dNTPase) that restricts HIV-1 by reducing cellular dNTP levels to below that required for reverse transcription. However, nothing is known about SAMHD1 posttranslational modifications and their potential role in regulating SAMHD1 function. We used (32)P labeling and immunoblotting with phospho-specific antibodies to identify SAMHD1 as a phosphoprotein. Several amino acids in SAMHD1 were identified to be sites of phosphorylation using direct mass spectrometry. Mutation of these residues to alanine to prevent phosphorylation or to glutamic acid to mimic phosphorylation had no effect on the nuclear localization of SAMHD1 or its sensitivity to Vpx-mediated degradation. Furthermore, neither alanine nor glutamic acid substitutions had a significant effect on SAMHD1 dNTPase activity in an in vitro assay. Interestingly, however, we found that a T592E mutation, mimicking constitutive phosphorylation at a main phosphorylation site, severely affected the ability of SAMHD1 to restrict HIV-1 in a U937 cell-based restriction assay. In contrast, a T592A mutant was still capable of restricting HIV-1. These results indicate that SAMHD1 phosphorylation may be a negative regulator of SAMHD1 restriction activity. This conclusion is supported by our finding that SAMHD1 is hyperphosphorylated in monocytoid THP-1 cells under nonrestrictive conditions.

Mycorrhizal responses in wheat: shading decreases growth but does not lower the contribution of the fungal phosphate uptake pathway.

Effects have been investigated of reduced C supply (induced by shade) on arbuscular mycorrhizal (AM) colonisation, mycorrhizal growth responses (MGRs) and on AM-mediated and direct uptake of phosphate (Pi) (using (32)P) in wheat, a plant that does not usually respond positively to AM colonisation. Shading markedly reduced growth and shoot/root dry weight ratios of both AM and non-mycorrhizal wheat, indicating decreased photosynthetic C supply. However, shading had very little effect on percent root length colonised by Rhizophagus irregularis or Gigaspora margarita or on MGRs, which remained slightly positive or zero, regardless of shade; there were no growth depressions under shade. By 6 weeks, when the contributions of the AM pathway were measured with (32)P supplied in small hyphal compartments, R. irregularis had supplied 23 to 28% of shoot P with no significant effect of shading. Data show that reduced C availability did not reduce the contribution of the AM pathway to plant P, so the fungi were not acting physiologically as parasites. These results support our previous hypothesis that lack of positive MGR is not necessarily the outcome of excessive C use by the fungi or failure to deliver P via the AM pathway.

Chlamydia trachomatis transports NAD via the Npt1 ATP/ADP translocase.

Obligate intracellular bacteria comprising the order Chlamydiales lack the ability to synthesize nucleotides de novo and must acquire these essential compounds from the cytosol of the host cell. The environmental protozoan endosymbiont Protochlamydia amoebophila UWE25 encodes five nucleotide transporters with specificities for different nucleotide substrates, including ATP, GTP, CTP, UTP, and NAD. In contrast, the human pathogen Chlamydia trachomatis encodes only two nucleotide transporters, the ATP/ADP translocase C. trachomatis Npt1 (Npt1(Ct)) and the nucleotide uniporter Npt2(Ct), which transports GTP, UTP, CTP, and ATP. The notable absence of a NAD transporter, coupled with the lack of alternative nucleotide transporters on the basis of bioinformatic analysis of multiple C. trachomatis genomes, led us to re-evaluate the previously characterized transport properties of Npt1(Ct). Using [adenylate-(32)P]NAD, we demonstrate that Npt1(Ct) expressed in Escherichia coli enables the transport of NAD with an apparent K(m) and V(max) of 1.7 µM and 5.8 nM mg(-1) h(-1), respectively. The K(m) for NAD transport is comparable to the K(m) for ATP transport of 2.2 µM, as evaluated in this study. Efflux and substrate competition assays demonstrate that NAD is a preferred substrate of Npt1(Ct) compared to ATP. These results suggest that during reductive evolution, the pathogenic chlamydiae lost individual nucleotide transporters, in contrast to their environmental endosymbiont relatives, without compromising their ability to obtain nucleotides from the host cytosol through relaxation of transport specificity. The novel properties of Npt1Ct and its conservation in chlamydiae make it a potential target for the development of antimicrobial compounds and a model for studying the evolution of transport specificity.

The interplay between P uptake pathways in mycorrhizal peas: a combined physiological and gene-silencing approach.

Arbuscular mycorrhizal fungi (AMF) have a key role in plant phosphate (Pi) uptake by their efficient capture of soil phosphorus (P) that is transferred to the plant via Pi transporters in the root cortical cells. The activity of this mycorrhizal Pi uptake pathway is often associated with downregulation of Pi transporter genes in the direct Pi uptake pathway. As the total Pi taken up by the plant is determined by the combined activity of mycorrhizal and direct pathways, it is important to understand the interplay between these, in particular the actual activity of the pathways. To study this interplay we modulated the delivery of Pi via the mycorrhizal pathway in Pisum sativum by two means: (1) Partial downregulation by virus-induced gene silencing of PsPT4, a putative Pi transporter gene in the mycorrhizal pathway. This resulted in decreased fungal development in roots and soil and led to reduced plant Pi uptake. (2) Changing the percentage of AMF-colonized root length by using non-, half-mycorrhizal or full-mycorrhizal split-root systems. The combination of split roots, use of ³²P and ³³P isotopes and partial silencing of PsPT4 enabled us to show that the expression of PsPT1, a putative Pi transporter gene in the direct pathway, was negatively correlated with increasing mycorrhizal uptake capacity of the plant, both locally and systemically. However, transcript changes in PsPT1 were not translated into corresponding, systemic changes in actual direct Pi uptake. Our results suggest that AMF have a limited long-distance impact on the direct pathway.

In situ UVA exposure modulates change in the uptake of radiophosphate in size-fractionated plankton assemblages following UVR exposure.

We investigated the effect of ultraviolet radiation (UVR) on the uptake and partitioning of radiophosphate ((33)PO (4) (3-) ) in size-fractionated plankton assemblages (0.2-0.8, 0.8-2.0 and >2.0 µm) collected from nine freshwater lakes located in Saskatchewan, Canada. A significant (p < 0.05) reduction in (33)PO (4) (3-) uptake by plankton was observed in seven of the nine lakes. Plankton >2.0 µm were generally unaffected by UVR, whereas the 0.2-0.8 µm size fraction exhibited severe photoinhibition. The effect of UVR on the 0.8-2.0 µm size fraction was variable, ranging from significant reductions to significant increases in (33)PO (4) (3-) uptake. The >2.0 µm size fraction was composed of a diversity of phytoplankton genera, suggesting that P uptake mechanisms for a range of phytoplankton are resistant to UVR. Our ability to detect a UVR effect on specific plankton size fractions was confounded by the resolution of the analysis. That is, only examining the <2.0 and >2.0 µm size fractions concealed the effect of UVR on plankton <0.8 µm. The magnitude of decrease in P uptake by plankton <0.8 µm was significantly and negatively correlated with in situ UVA exposure. Our results underscore the need for studies to consider both the size resolution of their analysis (i.e., the size of target organisms) and the ambient light conditions under which organisms may have acclimated before generalizing results across limnetic systems.

Activation of the abundant nuclear factor poly(ADP-ribose) polymerase-1 by Helicobacter pylori.

Modification of eukaryotic proteins is a powerful strategy used by pathogenic bacteria to modulate host cells during infection. Previously, we demonstrated that Helicobacter pylori modify an unidentified protein within mammalian cell lysates in a manner consistent with the action of a bacterial ADP-ribosylating toxin. Here, we identified the modified eukaryotic factor as the abundant nuclear factor poly(ADP-ribose) polymerase-1 (PARP-1), which is important in the pathologies of several disease states typically associated with chronic H. pylori infection. However, rather than being ADP-ribosylated by an H. pylori toxin, the intrinsic poly(ADP-ribosyl) polymerase activity of PARP-1 is activated by a heat- and protease-sensitive H. pylori factor, resulting in automodification of PARP-1 with polymers of poly(ADP-ribose) (PAR). Moreover, during infection of gastric epithelial cells, H. pylori induce intracellular PAR-production by a PARP-1-dependent mechanism. Activation of PARP-1 by a pathogenic bacterium represents a previously unrecognized strategy for modulating host cell signaling during infection.

Phosphorylation of Ser-80 of VP1 and Ser-254 of VP2 is essential for human BK virus propagation in tissue culture.

BK virus (BKV) infection may cause polyomavirus-associated nephropathy in patients with renal transplantation. Recently, the phosphorylated amino acids on the structural proteins VP1, VP2 and VP3 of BKV have been identified by liquid chromatography-tandem mass spectrometry in our laboratory. In this study, we further analysed the biological effects of these phosphorylation events. Phosphorylation of the BKV structural proteins was demonstrated by [(32)P]orthophosphate labelling in vivo. Site-directed mutagenesis was performed to replace all of the phosphorylated amino acids. The mutated BKV genomes were transfected into Vero cells for propagation analysis. The results showed that expression of the early protein LT and of the late protein VP1 by the mutants VP1-S80A, VP1-S80-133A, VP1-S80-327A, VP1-S80-133-327A and VP2-S254A was abolished. However, propagation of other mutants was similar to that of wild-type BKV. The results suggest that phosphorylation of Ser-80 of VP1 and Ser-254 of VP2 is crucial for BKV propagation.

Accumulation of intermediates of the carbon-phosphorus lyase pathway for phosphonate degradation in phn mutants of Escherichia coli.

The catabolism of phosphonic acids occurs in Escherichia coli by the carbon-phosphorus lyase pathway, which is governed by the 14-cistron phn operon. Here, several compounds are shown to accumulate in strains of E. coli with genetic blocks in various phn cistrons when the strains are fed with phosphonate.

Phosphotriester adducts (PTEs): DNA's overlooked lesion.

In addition to reacting with DNA base moieties, many chemical genotoxins also react with the oxygen atoms of the internucleotidic phosphodiester linkages to form phosphotriester adducts (PTEs). In view of their stability under physiological conditions, it has been suggested that PTEs may be useful biomarkers for measuring cumulative genotoxin exposure. The methodology for their determination is varied and still not completely developed but includes determination of hydrolysis products and (32)P-postlabelling approaches. More recently, transalkylation and direct mass spectrometry techniques have been devised, which give extra chemical information on the structures of the PTEs. The proportion of DNA damage formed as PTEs is much greater with SN1 compared to SN2 alkylating agents, and it has been shown in DNA that the formation of PTEs is partially sequence dependent. PTEs have been considered to be refractory to repair in mammalian cells but repair mechanisms have been found in prokaryotic cells, e.g. PTEs in Escherichia coli are repaired by O(6)-methylguanine-DNA methyltransferase (O(6)-MGT or Ada protein). However, studies on in vivo persistence of PTEs in mammalian systems have not ruled out the possibility of a contribution from an active repair process for PTEs. The biological significance of PTEs is largely unstudied and unknown, although effects of PTEs on DNA polymerases, and some exo- and endonucleases have been observed. Also site-specific PTEs impair the repair processing of adjacent sites of DNA damage, which may be a biological mechanism of importance for these lesions. In this review, we will consider the analytical methods available for the determination of PTEs, their stability in vitro and in vivo, the mechanisms for their repair, their possible biological significance and their potential role as biomarkers in human molecular epidemiology studies.

Labeling 3' Termini of Double-Stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I.

The Klenow fragment, which retains the template-dependent deoxynucleotide polymerizing activity and the 3' → 5' exonuclease of the holo-enzyme but lacks its powerful 5' → 3' exonuclease activity, is used to fill recessed 3' termini of dsDNA. In this protocol, fragments suitable as templates for the end-filling reaction are produced by digestion of DNA with an appropriate restriction enzyme. The Klenow enzyme is then used to catalyze the attachment of dNTPs to the recessed 3'-hydroxyl groups.

The CBS subdomain of inosine 5'-monophosphate dehydrogenase regulates purine nucleotide turnover.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyses the rate-limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP content was substantially elevated in MP101 whereas the GTP content was slighty reduced. The activities of IMPDH, adenylosuccinate synthetase and GMP reductase were two to threefold lower in MP101 crude extracts compared with the BW25113 wild-type strain. Guanine induced a threefold reduction in the MP101 ATP pool and a fourfold increase in the GTP pool within 10 min of addition to growing cells; this response does not result from the reduced IMPDH activity or starvation for guanylates. In vivo kinetic analysis using 14-C tracers and 33-P pulse-chasing revealed mutation-associated changes in purine nucleotide fluxes and turnover rates. We conclude that the CBS subdomain of IMPDH may coordinate the activities of the enzymes of purine nucleotide metabolism and is essential for maintaining the normal ATP and GTP pool sizes in E. coli.

Functional and structural dynamics of hepadnavirus reverse transcriptase during protein-primed initiation of reverse transcription: effects of metal ions.

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed epsilon. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg(2+). Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn(2+), in comparison with Mg(2+), showed dramatic effects on the priming activity of MiniRT2 as well as the full-length RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn(2+), indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg(2+). Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn(2+). Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn(2+). Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn(2+) but not in the presence of Mg(2+). Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn(2+) versus that of Mg(2+) and that the transition from initiation to polymerization was accompanied by RT conformational change.

Systematic study of the functions for the residues around the nucleotide pocket in simian virus 40 AAA+ hexameric helicase.

The high-resolution structural data for simian virus 40 large-T-antigen helicase revealed a set of nine residues bound to ATP/ADP directly or indirectly. The functional role of each of these residues in ATP hydrolysis and also the helicase function of this AAA+ (ATPases associated with various cellular activities) molecular motor are unclear. Here, we report our mutational analysis of each of these residues to examine their functionality in oligomerization, DNA binding, ATP hydrolysis, and double-stranded DNA (dsDNA) unwinding. All mutants were capable of oligomerization in the presence of ATP and could bind single-stranded DNA and dsDNA. ATP hydrolysis was substantially reduced for proteins with mutations of residues making direct contact with the gamma-phosphate of ATP or the apical water molecule. A potentially noncanonical "arginine finger" residue, K418, is critical for ATP hydrolysis and helicase function, suggesting a new type of arginine finger role by a lysine in the stabilization of the transition state during ATP hydrolysis. Interestingly, our mutational data suggest that the positive- and negative-charge interactions in the uniquely observed residue pairs, R498/D499 and R540/D502, in large-T-antigen helicase are critically involved in the transfer of energy of ATP binding/hydrolysis to DNA unwinding.

Invasive plants versus their new and old neighbors: a mechanism for exotic invasion.

Invading exotic plants are thought to succeed primarily because they have escaped their natural enemies, not because of novel interactions with their new neighbors. However, we find that Centaurea diffusa, a noxious weed in North America, has much stronger negative effects on grass species from North America than on closely related grass species from communities to which Centaurea is native. Centaurea's advantage against North American species appears to be due to differences in the effects of its root exudates and how these root exudates affect competition for resources. Our results may help to explain why some exotic species so successfully invade natural plant communities.

Molecular neurodevelopment: an in vivo 31P-1H MRSI study.

Synaptic development and elimination are normal neurodevelopmental processes, which if altered could contribute to various neuropsychiatric disorders. 31P-1H magnetic resonance spectroscopic imaging (MRSI) and structural magnetic resonance imaging (MRI) exams were conducted on 105 healthy children ages 6-18 years old to identify neuromolecular indices of synaptic development and elimination. Over the age range studied, age-related changes in high-energy phosphate (phosphocreatine), membrane phospholipid metabolism (precursors and breakdown products), and percent gray matter volume were found. These neuromolecular and structural indices of synaptic development and elimination are associated with development of several cognitive domains. Monitoring of these molecular markers is essential for devising treatment strategies for neurodevelopmental disorders.

Measurements of 18 O-Pi uptake indicate fast metabolism of phosphate in tree roots.

Phosphorus (P) nutrition of beech ecosystems depends on soil processes, plant internal P cycling and P acquisition. P uptake of trees in the field is currently not validated due to the lack of an experimental approach applicable in natural forests. Application of radiolabelled tracers such as 33 P and 32 P is limited to special research sites and not allowed in natural environments. Moreover, only one stable isotope of P, namely 31 P, exists. One alternative tool to measure P acquisition in the field could be the use of 18 O-labelled 31 P-phosphate (31 P18 O4 3- ). Phosphate (Pi ) uptake rates calculated from the 18 O enrichment of dried root material after application of 31 Pi 18 O4 3- via nutrient solution was always lower compared to 33 P incorporation, did not show increasing rates of Pi uptake at P deficiency under controlled conditions, and did not reveal seasonal fluctuations in the field. Consequently, a clear correlation between 33 P-based and 18 O-based Pi uptake by roots could not be established. Comparison of Pi  uptake rates achieved from 33 P-Pi and 18 O-Pi application led to the conclusion of high Pi metabolism in roots after Pi uptake. The replacement of 18 O by 16 O from water in 18 O-Pi during root influx, but most probably after Pi uptake into roots, due to metabolic activities, indicates high and fast turnover of Pi . Hence, the use of 18 O-Pi as an alternative tool to estimate Pi acquisition of trees in the field must consider the increase of 18 O abundance in root water that was disregarded in dried root material.

Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers.

This protocol describes the generation of radiolabeled cDNA probes from poly(A)+ RNA in a random priming reaction. Probes of this type are used for differential screening of cDNA libraries.

Unexpected phosphoryl transfer from Asp351 to fluorescein attached to Lys515 in sarcoplasmic reticulum Ca2+-ATPase.

Sarcoplasmic reticulum Ca(2+)-ATPase is an ion pump whose catalytic cycle includes the transient formation of an acyl phosphate at Asp(351), and fluorescein isothiocyanate is a covalent inhibitor of ATP binding to this pump, known to specifically derivatize Lys(515) in the nucleotide-binding site. It was previously found that an unusually stable, phosphorylated form of fluorescein-ATPase, with low fluorescence, is obtained following Ca (2+) loading with acetyl phosphate as energy source and then chelation with EGTA of Ca(2+) on the cytosolic side. Here we show that the phospho-linkage in this low fluorescent species is stable at alkaline pH, unlike the acyl phosphate at Asp(351). Moreover, the low fluorescence and stable phosphoryl group track together in primary and secondary tryptic subfragments, separated by SDS-PAGE after denaturation. Finally, normal fluorescence and absorbance are recovered upon treatment with alkaline phosphatase after extensive trypsinolysis. We conclude that the low fluorescent species is the result of the phosphoryl group being transferred from Asp (351) to the fluorescein moiety during pump reversal, yielding fluorescein monophosphate tethered to Ca(2+)-ATPase.