Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.     

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.     

Glutamine synthetase degradation is controlled by oxidative proteolysis in the marine cyanobacterium Prochlorococcus marinus strain PCC 9511

Prochlorococcus is one of the most important primary producers on Earth; its unusual features and ecological importance have made it a model organism, but nutrient assimilation has received little attention. Glutamine synthetase (GS) plays a key role in nitrogen metabolism and its central position justifies the fine regulation of this enzyme. The aim of this work is to demonstrate the involvement of metal-catalyzed oxidation (MCO) in the control of the biological activity and turnover of GS from Prochlorococcus. In order to study the physiological role of MCO, we have first characterized the in vitro biosynthetic inactivation and degradation of GS in the axenic PCC 9511 strain, testing then the effect of several stress conditions, such as the presence of electron transport inhibitors, darkness and aging, on the inactivation and degradation of GS. It is noteworthy that the physiological substrates of GS could protect the enzyme from the oxidative inactivation and ATP partially reverted this inactivation once the enzyme had been oxidized, being this effect higher in the presence of glutamate. We have also found that the GS from aged cultures is degraded to the same smaller size fragments obtained in the in vitro degradation of GS by an oxidative model system (Fe3+/NADH/NADH oxidase/O2). These results suggest the implication of MCO in the age- and oxidative state-dependent degradation of GS from Prochlorococcus.     

In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with RRNA-targeted peptide nucleic acid probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.     

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C₂H₂-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2–3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.Abbreviation GS glutamine synthetase     

Isolation and characterization of Photosystem I from two strains of the marine oxychlorobacterium Prochlorococcus

Photosystem I (PS I) complexes from two strains of the marine photosynthetic prokaryote Prochlorococcus, MED4 (= clone CCMP1378) and SS120 (= clone CCMP1375), were isolated by centrifugation on sucrose gradients after detergent treatment. The PS I-enriched fractions of both strains contained about 100 chlorophyll molecules per P700. Electron microscopy showed that the PS I complexes were in a trimeric form. The characteristic long wavelength fluorescence emission of PS I at 77 K, currently observed in chloroplasts and most cyanobacteria was absent both in intact cells and in PS I preparations of both strains. The major proteins of the PS I-enriched fractions were identified immunologically as PsaA and PsaB. Two proteins with apparent molecular masses of about 21 and 25 kDa were present in PS I preparations of Prochlorococcus, whereas the small PS I subunits in cyanobacteria all have molecular masses below 18 kDa. The 25 kDa protein showed a strong cross-reaction with a heterologous antibody against PsaL. Relatedness of the 21 kDa protein to PsaF was demonstrated by internal protein sequencing. Although only trace amounts of the major divinyl-Chl a/b-binding antenna complexes were present in the PS I preparations, significant amounts of divinyl-Chl b were observed in this fraction. The putative organization of this Chl b in PS I is discussed.     

Glutamine synthetase and glutamine synthetase-like protein from human brain: purification and comparative characterization

Glutamine synthetase (GS; EC 6.3.1.2), a key enzyme of glutamate metabolism, and another enzyme possessing high hydroxylamine-L-glutamine transferase activity comparable to that of GS and termed GS-like protein (GSLP) were purified from human brain concurrently. In two-dimensional electrophoresis, GS subunits migrate to at least six different positions (44 +/- 1 kDa, pl = 6. 4-6.7), whereas GSLP subunits migrate to at least four different positions (54 +/- 1 kDa, pl = 5.9-6.2). Dependences of enzymatic activity in the transferase reaction on concentrations of Mn(2+) and Mg(2+) for GS and GSLP are different. High immunological cross-reactivity between GS and GSLP was observed in ELISA. Nevertheless, antisera were raised to GS and GSLP, and a method was developed for the separate detection of GS and GSLP in brain extracts by enzyme-chemiluminescent amplified (ECL) immunoblotting. The distribution of GS and GSLP immunoreactivities between soluble protein and crude mitochondrial fractions indicates tighter association with the particulate fraction for GSLP than for GS. The results from activity measurements suggest that the hydroxylamine-L-glutamine transferase activity measured routinely in protein extracts from brain is the sum of GS and GSLP activities. Similarly, immunoreactivity evaluated by ELISA is a sum of immunoreactivities of GS and GSLP. The relative contributions of GS and GSLP to the total immunoreactivity can be evaluated by ECL-immunoblotting.     

Glutamine synthetase from a cyanobacterium, Phormidium lapideum: purification, characterization, and comparison with other cyanobacterial enzymes

Glutamine synthetase has been purified to homogeneity from cell extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The subunit molecular weight of the enzyme was determined as about 59,000 by sodium dodecyl sulfate gel electrophoresis. Electron micrographs of the Phormidium enzyme revealed a two-layered structure of regular hexagons (12 subunits per molecule), which markedly resembles the three-dimensional polypeptide backbone structure of the Salmonella typhimurium glutamine synthetase established by X-ray crystallography (Almassy, Janson, Hamlin, Xuong, & Eisenberg (1986) Nature 323, 304-309). The N-terminal amino acid sequence of the Phormidium enzyme shows very high similarity with that of the enzyme from an N2-fixing cyanobacterium, Anabaena 7120; 18 residues are common in 23 residues compared. Strong immunocross-reactions between the antibody against the purified Phormidium glutamine synthetase and other cyanobacterial enzymes except the Anacystis enzyme were observed. The apparent Michaelis constants for NH3, L-glutamate, and ATP were determined to be 0.29, 7.4, and 1.7 mM, respectively. Divalent metal ions such as Mg2+ and Mn2+ activated the enzyme in the biosynthetic reaction, whereas various amino acids and glutamate analogs strongly inhibited the enzyme.     

Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation

Aspartokinase II from Bacillus subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients. Ammonium starvation induced the fastest aspartokinase II decline (t1/2 = 65 min), followed by amino acid starvation (t1/2 = 80 min) and glucose limitation (t1/2 = 120 min). Loss of enzyme activity was closely correlated with the disappearance of the alpha subunit; degradation of the beta subunit was somewhat delayed or slower under some conditions. Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion. The degradation of aspartokinase II was interrupted by inhibitors of energy production and protein synthesis but was not changed in a mutant lacking a major intracellular protease. Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells. These results indicate that although energy-dependent degradation of biosynthetic enzymes is a general phenomenon in nutrient-starved B. subtilis cells, the degradation of specific enzymes probably involves different pathways.     

Glutamine synthetase of Dunaliella primolecta. Partial characterization and possible adenylation control in relation to nitrogen nutrient levels

Glutamine synthetase (GS, E.C. 6.3.1.2.) of the unicellular alga Dunaliella primolecta has been partially purified by gel filtration and affinity chromatography. The molecular weight of the enzyme has been estimated at 480,000, comprising eight subunits of 60,000 each. The kinetic behaviour of the enzyme exhibits a biphasic profile of substrate saturation, corresponding to a negative cooperativity process. Alanine, carbamoyl phosphate and glucosamine exert a strong inhibitory effect. The feedback control is cumulative. The effect of Mn²⁺ and Mg²⁺ has been studied. The results suggest the existence of an adenylation process and the possibility of a role of Dunaliella GS in the overall control of nitrogen assimilation.     

Proteomic response of the marine ammonia-oxidising archaeon Nitrosopumilus maritimus to iron limitation reveals strategies to compensate for nutrient scarcity

Dissolved iron (Fe) is vanishingly low in the oceans, with ecological success conferred to microorganisms that can restructure their biochemistry to maintain high growth rates during Fe scarcity. Chemolithoautotrophic ammonia-oxidising archaea (AOA) are highly abundant in the oceans, constituting ~30% of cells below the photic zone. Here we examine the proteomic response of the AOA isolate Nitrosopumilus maritimus to growth-limiting Fe concentrations. Under Fe limitation, we observed a significant reduction in the intensity of Fe-dense ferredoxins associated with respiratory complex I whilst complex III and IV proteins with more central roles in the electron transport chain remain unchanged. We concomitantly observed an increase in the intensity of Fe-free functional alternatives such as flavodoxin and plastocyanin, thioredoxin and alkyl hydroperoxide which are known to mediate electron transport and reactive oxygen species detoxification, respectively. Under Fe limitation, we found a marked increase in the intensity of the ABC phosphonate transport system (Phn), highlighting an intriguing link between Fe and P cycling in N. maritimus. We hypothesise that an elevated uptake of exogenous phosphonates under Fe limitation may either supplement N. maritimus' endogenous methylphosphonate biosynthesis pathway - which requires Fe - or enhance the production of phosphonate-containing exopolysaccharides known to efficiently bind environmental Fe.     

Glutamine synthetase. IX. Purification and characterization of the enzyme from sheep spleen.

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) has been purified about 550-fold from sheep spleen. The subunit weight of the enzyme is estimated to be 48 000. Sedimentation coefficient determination by density gradient centrifugation gives a value of 15.0 S. The approximate molecular weight calculated from the S value is 378500. In addition, electron micrographs of the enzyme show an "H" shape. Hence, the protein appears to have eight subunits. In sheep spleen, the enzyme resides chiefly in the soluble fraction of the cell. The amino acid composition of the enzyme from spleen shows similarity to that from other sources. The enzyme activity is nearly five times as high in Mg2+ as in Mn2+. ATP inhibits the enzyme; the inhibition is competitive with respect to Mg2+ATP. A number of compounds, such as D-alanine, AMP, creatine phosphate, arsenite in combination with 2,3-dimercaptopropanol, and 2-amino-4-phosphonobutyrate, also inhibit the enzyme. The inhibition by the last compound is competitive with respect to glutamate. D-Glutamate and alpha-methyl-DL-glutamate can serve as substrates in the synthesis reaction, but N-methyl-DL-glutamate cannot. On the other hand, neither D-glutamine nor N-acetyl-L-glutamine can replace L-glutamine as a substrate in the gamma-glutamyl transfer reaction of the enzyme. Inhibition of Mn2+ and ATP and its reversal by Mg2+ have been discussed as a means of regulating the enzyme activity in mammalian tissues.     

Bioenergetics and end-product regulation of Clostridium thermosaccharolyticum in response to nutrient limitation

Fermentation of xylose by Clostridium thermosaccharolyticum was studied in batch and continuous culture in which the limiting nutrient was either xylose, phosphate, or ammonia. Transient results obtained in continuous cultures with batch grown inoculum and progressively higher feed substrate concentrations exhibited ethanol selectivities (moles ethanol/moles other products) in excess of 11. The hypothesis that this high ethanol selectivity was a general response to mineral nutrient limitation was tested but could not be supported. Growth and substrate consumption were related by the equation q(s)(1 - Y(x) (c))G(ATP) = (mu/Y(ATP) (max)) + m, with q(s) the specific rate of xylose consumption (moles xylose/hour . g cells), Y(x) (c) the carbon based cell yield (g cell carbon/g substrate carbon), G(ATP) the ATP gain (moles ATP produces/mol substrate catabolized), mu the specific growth rate (1/h), Y(ATP) (max) the ATP-based cell yield (g cells/mol ATP), and m the maintenance coefficient (moles ATP/hour . g cells). Y(ATP) (max) was found to be 11.6 g cells/mol ATP, and m 9.3 mol ATP/hour . g cells for growth on defined medium. Different responses to nutrient limitation were observed depending on the mode of cultivation. Batch and immobilized cell continuous cultures decreased G(ATP) by initiating production of the secondary metabolites, propanediol, and in some cases, D-lactate; in addition, batch cultures increased the fractional allocation of ATP to maintenance and/or wastage. Nitrogen-limited continuous free-cell cultures maintained a constant cell yield, whereas phosphate-limited continuous free-cell cultures did not. In the case of phosphate limitation, the decreased ATP demand associated with the lowered cell yield was accompanied by an increased rate of ATP consumption for maintenance and/or wastage. Neither nitrogen or phosphorus-limited continuous free-cell cultures exhibited an altered G(ATP) in response to mineral nutrient limitation, and neither produced secondary metabolites. (c) 1993 John Wiley & Sons, Inc.     

Morphological, chemical, and genetic diversity of tropical marine cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales)

Although diverse natural products have been isolated from the benthic, filamentous cyanobacterium Lyngbya majuscula, it is unclear whether this chemical variation can be used to establish taxonomic relationships among disparate collections. We compared morphological characteristics, secondary-metabolite compositions, and partial 16S ribosomal DNA (rDNA) sequences among several collections of L. majuscula Gomont, Lyngbya spp., and Symploca spp. from Guam and the Republic of Palau. The morphological characteristics examined were cell length, cell width, and the presence or absence of a calyptra. Secondary metabolites were analyzed by two-dimensional thin-layer chromatography. Each collection possessed a distinct cellular morphology that readily distinguished Lyngbya spp. from Symploca spp. Each collection yielded a unique chemotype, but common chemical characteristics were shared among four collections of L. majuscula. A phylogeny based on secondary-metabolite composition supported the reciprocal monophyly of Lyngbya and Symploca but yielded a basal polytomy for Lyngbya. Pairwise sequence divergence among species ranged from 10 to 14% across 605 bp of 16S rDNA, while collections of L. majuscula showed 0 to 1.3% divergence. Although the phylogeny of 16S rDNA sequences strongly supported the reciprocal monophyly of Lyngbya and Symploca as well as the monophyly of Lyngbya bouillonii and L. majuscula, genetic divergence was not correlated with chemical and morphological differences. These data suggest that 16S rDNA sequence analyses do not predict chemical variability among Lyngbya species. Other mechanisms, including higher rates of evolution for biosynthetic genes, horizontal gene transfer, and interactions between different genotypes and environmental conditions, may play important roles in generating qualitative and quantitative chemical variation within and among Lyngbya species.     

Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum.

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.     

Low-molecular mass carbohydrate accumulation in cyanobacteria from a marine microbial mat in response to salt

Forty seven strains of cyanobacteria, all isolated from microbial mats of intertidal sediments of the island of Mellum (North Sea), were analyzed for the presence of organic osmotica. The cyanobacteria examined belonged to taxonomically different groups and were classified according to their salt optimum and salt tolerance as either freshwater, brackish or marine. Except betaine, all organic osmotica known to occur in cyanobacteria, were found. The results showed no clear correlation between the chemical nature of the organic solute and the salt optimum or salt tolerance of the cyanobacteria examined, indicating that these solutes are not specific to this marine habitat. All strains belonging to the Nostoc/Anabea-group accumulated sucrose as the sole organic osmoticum. The marine, heterocystous Calothrix sp. accumulated trehalose. All strains of the LPP-group (Lyngbya, Plectonema, Phormidium) accumulated glucosylglycerol as sole or primary organic solute. Some LPP-strains accumulated a disaccharide as a secundary solute, e.g. sucrose or trehalose. Gloeocapsa, Synechocystis and Spirulina accumulated glucosylglycerol. Two marine Oscillatoria accumulated trehalose, whereas a freshwater Oscillatoria with a broad salinity tolerance, accumulated sucrose.Analysis of field samples of the microbial mats demonstrated the presence of glycerol, glucosylglycerol, sucrose and trehalose. The relative abundance of the different compounds was related to the species composition as could be predicted from laboratory observations. These data suggest that these carbohydrates have a function in maintaining osmotic balance in the organisms within the microbial mat.     

Seed size and seedling growth: differential response of Australian and British Fabaceae to nutrient limitation

Seed size is widely held to exert an important influence over plant establishment, but while large seeds are often assumed to be at an advantage in nutrient-limited conditions, there is in fact, little consistent evidence to support this hypothesis. Here, we examined the interspecific relationship between seedling growth and seed size for Australian and British Fabaceae species in nutrient solutions deficient in nitrogen, phosphorus, potassium or all nutrients combined (distilled water). The British species showed no consistent link between mean seed mass and seedling growth in nutrient-limited conditions. By contrast, all four nutrient-deficient treatments yielded a significant relationship for the Australian species. Linear regression showed that growth under balanced nutrient conditions was positively associated with growth without nutrients, although in fewer cases for the British species. We suggest that habitat-specific differences in regeneration conditions and/or evolutionary history may influence the role that seed size plays in dictating how seedlings of different species respond to nutrient shortage. We recommend caution in attempts to link traits like seed size to wider patterns of plant community ecology.     

Molecular phylogeny and surface morphology of marine aseptate gregarines (Apicomplexa): Selenidium spp. and Lecudina spp

Many aseptate gregarines from marine invertebrate hosts are thought to have retained several plesiomorphic characteristics and are instrumental in understanding the early evolution of intracellular parasitism in apicomplexans and the phylogenetic position of cryptosporidians. We sequenced the small-subunit (SSU) ribosomal RNA genes from 2 archigregarines, Selenidium terebellae and Selenidium vivax, and 2 morphotypes of the marine eugregarine Lecudina polymorpha. We also used scanning electron microscopy to investigate the surface morphology of trophozoites from Lecudina tuzetae, Monocystis agilis, the 2 species of Selenidium, and the 2 morphotypes of L. polymorpha. The SSU ribosomal DNA sequences from S. vivax and L. polymorpha had long branch lengths characteristic of other gregarine sequences. However, the sequence from S. terebellae was not exceptionally divergent and consistently emerged as 1 of the earliest 'true' gregarines in phylogenetic analyses. Statistical support for the sister relationship between Cryptosporidium spp. and gregarines was significantly bolstered in analyses including the sequence from S. terebellae but excluding the longest branches in the alignment. Eugregarines formed a monophyletic group with the neogregarine Ophryocystis, suggesting that trophozoites with elaborate cortex folds and gliding motility evolved only once. The trophozoites from the 2 species of Selenidium shared novel transverse striations but differed from one another in overall cell morphologies and writhing behavior.