Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast

Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low‐molecular‐weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)₃ was also detected and direct transport assays demonstrate that As(GS)₃ does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.     

Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis

Summary Ethylenediamine (EDA) is toxic to the cyanobacterium Anabaena variabilis and inhibits nitrogenase activity. The inhibition of nitrogenase was prevented by pretreatment of cells with l-methionine-d,l-sulphoximine (MSX). Mutant strains of Anabaena variabilis (ED81, ED92), resistant to EDA, had low levels of glutamine synthetase (GS) biosynthetic activity compared with the wild type strain. ED92 had a low level of GS protein whereas ED81 had a similar level to that of the parent strain as estimated using antibodies against GS. Both strains fixed N₂ and liberated NH₄ ⁺ into the media. Following immobilization of the mutant strains, sustained photoproduction of NH₄ ⁺ was obtained in air-lift reactors at rates of up to 50 mol NH₄ ⁺ mg chl a^–1 h^–1, which were comparable to the rates obtained when immobilized cyanobacteria were treated with MSX.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - MSX l-methionine-d,l-sulphoximine     

Arsenite oxidizing multiple metal resistant bacteria isolated from industrial effluent: their potential use in wastewater treatment

Arsenite oxidizing bacteria, isolated from industrial wastewater, showed high resistance against arsenite (40 mM) and other heavy metals (10 mM Pb; 8 mM Cd; 6 mM Cr; 10 mM Cu and 26.6 mM As⁵⁺). Bacterial isolates were characterized, on the basis of morphological, biochemical and 16S rRNA ribotyping, as Bacillus cereus (1.1S) and Acinetobacter junii (1.3S). The optimum temperature and pH for the growth of both strains were found to be 37 °C and 7. Both the strains showed maximum growth after 24 h of incubation. The predominant form of arsenite oxidase was extracellular in B. cereus while in A. junii both types of activities, intracellular and extracellular, were found_. The extracellular aresenite oxidase activity was found to be 730 and 750 µM/m for B. cereus and A. junii, respectively. The arsenite oxidase from both bacterial strains showed maximum activity at 37 °C, pH 7 and enhanced in the presence of Zn²⁺. The presence of two protein bands with molecular weight of approximately 70 and 14 kDa in the presence of arsenic points out a possible role in arsenite oxidation. Arsenite oxidation potential of B. cereus and A. junii was determined up to 92 and 88 % in industrial wastewater after 6 days of incubation. The bacterial treated wastewater improved the growth of Vigna radiata as compared to the untreated wastewater. It indicates that these bacterial strains may find some potential applications in wastewater treatment systems to transform toxic arsenite into less toxic form, arsenate.     

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag⁺, AsO ⁻₂ , AsO ³⁻₄ , Cd²⁺, Co²⁺, CrO ²⁻₄ , Cu²⁺, Hg²⁺, Ni²⁺, Pb²⁺, TeO ²⁻₃ , Tl⁺ and Zn²⁺. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd²⁺-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd²⁺, Zn²⁺, and Co²), others are known that efflux Ag⁺, Cu⁺, Ni²⁺, and Zn²⁺. Resistance to inorganic mercury, Hg²⁺ (and to organomercurials, such as CH₃Hg⁺ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.

     

Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.     

Decomposition of four macrophytes in wetland sediments: Organic matter and nutrient decay and associated benthic processes

Decomposition rates of Phragmites australis, Carex riparia, Nuphar luteum and Salvinia natans and benthic processes were measured from December 2003 to December 2004 in a shallow wetland (Paludi di Ostiglia, Northern Italy) by means of litter bags and intact cores incubations. Decay rate was highest for N. luteum (k = 0.0152 d⁻¹), intermediate for S. natans (k = 0.0041 d⁻¹) and similar for P. australis (k = 0.0027 d⁻¹) and C. riparia (k = 0.0028 d⁻¹).Benthic metabolism followed a seasonal pattern with summer peaks of O₂ demand and TCO₂, CH₄ and NH₄⁺ efflux whilst soluble reactive phosphorus (SRP) fluxes were negligible also under hypoxic conditions, indicating that P was mainly retained by sediment. The initial C:P ratio was similar in N. luteum and S. natans (170) and significantly lower than that of P. australis and C. riparia (360). During the detritus decay P was progressively lost by N. luteum and S. natans tissues, whereas, after an initial leaching, it was probably re-used during the microbial decomposition of the more refractory P. australis and C. riparia detritus. Nuphar luteum, P. australis and S. natans had comparable initial C:N mass ratio (15), significantly lower than that of C. riparia (26). The C:N ratio was rather constant for N. luteum (12.9 ± 1.5) and S. natans (14.6 ± 0.9), decreased slightly to below 20 for C. riparia and increased up to 30 for P. australis. Overall, differences among species were likely due to the recalcitrance of decomposing detritus, whilst process rates were controlled by limitation of microbial processes by nutrients and electron acceptor availability.     

Arsenic exposure alters activity behaviour of key nitrogen assimilatory enzymes in growing rice plants

Rice seedlings when grown in sand cultures for 5–20 days under 25 and 50 M As₂O₃ in the medium showed a marked decline in growth when compared to controls. Increased absorption of arsenic from the medium, against the concentration gradient was observed. Greater localization of absorbed arsenic was noted in roots than in shoots. Rice plants grown for 20 days with 50 mol l^–1 arsenic in the medium accumulated upto 370 mol arsenic kg^–1 dry weight in roots. Increasing levels of As₂O₃ in situ caused a marked decline in the activities of the nitrate assimilatory enzymes nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS), whereas an increase in the activities of alanine and aspartate aminotransferases was observed. The activities of aminating (NADH-GDH) and deaminating (NAD⁺-GDH) glutamate dehydrogenases increased at moderately toxic level (25 M) of As₂O₃ whereas a higher As level of 50 M was inhibitory to the enzymes. Addition of 1 M proline in the reaction medium caused significant restoration in As-led loss of NR and GS activities. NR and GS extracted from arsenic exposed seedlings showed higher K _m values compared to the enzymes extracted from control-grown seedlings, whereas GDHs extracted from As-stressed seedlings showed a decrease in K _m. Results suggest that inhibition in the activities of N assimilatory enzymes accompanied with decreased affinity of the enzymes towards their substrates would eventually lead to a marked suppression of N assimilation and impaired growth of rice seedlings in As polluted environment.     

The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa

Summary Growth of Neurospora crassa on media containing NH ₄ ⁺ leads to the repression of a variety of permeases and alternative pathways which would generate NH ₄ ⁺ , so called ammonium repression. The mutant am ₂ which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-la has derepressed levels of the aforementioned systems unless grown with glutamine.The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am ₂ and gln-la when they are ammonium derepressed.The mechanism of NH ₄ ⁺ repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH ₄ ⁺ sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.     

Dehalogenation of chlorinated aromatic compounds using a hybrid bioinorganic catalyst on cells of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis>

A novel bioinorganic catalyst was obtained via reduction of Pd(II) to Pd⁰ on to the surface of cells of Desulfovibrio desulfuricans at the expense of H₂. Palladised biomass, supplied with formate or H₂ as an electron donor, catalysed the dehalogenation of 2-chlorophenol and polychlorinated biphenyls. In the example of 2,3,4,5-tetrachlorobiphenyl, the bioinorganic catalyst promoted a rate of chloride release of 9.33 ± 0.17 nmol min^–1 mg ^–1and only ~5% of this value was obtained using chemically reduced or commercially available Pd ⁰. In the case of 2,2,4,4,6,6-hexachlorobiphenyl the rate was more than four orders of magnitude faster than the degradation reported using a sulfidogenic culture. Negligible chloride release occurred from any of the chloroaromatic compounds using biomass alone, or from palladised biomass challenged with hexane carrier solvent only. Analysis of the spent solution showed that in addition to catalysis of reductive dehalogenation the new material was able to remove very effectively the organic residua, with neither any PCB nor any breakdown products identifiable by GC/MS.Revisions requested 8 September 2004; Revisions received 21 October 2004;     

Synthesis of novel ketoconazole derivatives as inhibitors of the human Pregnane X Receptor (PXR; NR1I2; also termed SXR, PAR)

PXR, pregnane X receptor, in its activated state, is a validated target for controlling certain drug–drug interactions in humans. In this context, there is a paucity of inhibitors directed toward activated PXR. Using prior observations with ketoconazole as a PXR inhibitor, the target compound 3 was synthesized from (s)-glycidol with overall 56% yield. (+)-Glycidol was reacted with 4-bromophenol and potassium carbonate in DMF to yield the ring opened compound 6. This was then heated to reflux in benzene along with 2′, 4′-difluoroacetophenone and catalytic amount of para-toluene sulfonic acid to yield 8. The resultant acetal 8 was then functionalized using Palladium chemistry to yield the target compound 3. The activity of the compound was compared with ketoconazole and UCL2158H. However, in contrast with ketoconazole (IC₅₀  0.020 μM; 100% inhibition), 3 has negligible effects on inhibition of microsomal CYP450 (maximum 20% inhibition) at concentrations >40 μM. In vitro, micromolar concentration of ketoconazole is toxic to passaged human cell lines, while 3 does not exhibit cytotoxicity up to concentrations 100 μM (viability >85%). This is the first demonstration of a chemical analog of a PXR inhibitor that retains activity against activated PXR. Furthermore, in contrast with ketoconazole, 3 is less toxic in human cell lines and has negligible CYP450 activity.     

Covalent modification of bacterial glutamine synthetase: physiological significance

Summary Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH₄ ⁺). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to 10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH₄ ⁺ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH₄ ⁺-excess conditions and thereby to allow rapid growth.     

Western equatorial Pacific planktic foraminiferal fluxes and assemblages during a La Niña year (1999)

A correlation between foraminiferal community dynamics and environmental conditions may provide a basis for establishing paleoclimatic proxies. We studied planktic foraminiferal shell fluxes and assemblages in samples collected in three time-series sediment trap deployments in the western equatorial Pacific under La Niña conditions from January to November 1999. Eleven species contributed about 90% of the total flux in all traps. Two sites (MT1, MT3) in the Western Pacific Warm Pool region (WPWP) were characterized by common occurrences of the species Globigerinoides ruber, Globigerinoides sacculifer, Globigerinoides tenellus, and Neogloboquadrina dutertrei. Site MT5 farther to the east in the equatorial upwelling region had common occurrences of Globigerina bulloides, Globigerinita glutinata, and Pulleniatina obliquiloculata. Very high abundances of G. bulloides and G. glutinata at MT5 indicate that equatorial upwelling (EU) occurred during the 1999 La Niña. The two western sites have similar assemblage compositions, but MT1 ( 135°E) has the highest fluxes (up to  3800 tests m^− 2 day^− 1), whereas MT3 ( 145° E) has fluxes below  2200 tests m^− 2 day^− 1. Relatively high fluxes (up to  3000 tests m^− 2 day^− 1) occur at site MT5 ( 176° E), where upwelling occurred.The differences in faunal composition in the WPWP and EU might be attributable to differences in the way in which nutrients are supplied to the phytoplankton: large amounts of suspended material are supplied to the WPWP by advection of waters passing through the coastal region of an archipelago, whereas upwelling of nutrient-rich waters enhances primary production in the EU. At the westernmost site in the WPWP, a peak in the G. bulloides flux coincided with southward flow of the New Guinea Coastal Current (NGCC) in late February, but the highest G. ruber flux coincided with northward flow of this current in late May. Thus, the differences in species dominance at this location may be caused by monsoon-driven variability in the flow direction of the NGGC.     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.)

The -amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO ₃ ^– nutrition) and also in the lateral roots (NH ₄ ⁺ nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed -mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO ₃ ^– -fed plants) or even increased (NH ₄ ⁺ -fed plants).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Organ-specific changes in the activity and subunit composition of glutamine-synthetase isoforms of barley (Hordeum vulgare L.) after growth on different levels of NH+ 4

One cytosolic glutamine synthetase (GS, EC 6.3.1.2) isoform (GS 1a) was active in the germinating seeds of barley (Hordeum vulgare L.). A second cytosolic GS isoform (GS 1b) was separated from the leaves as well as the roots of 10-d-old seedlings. The chloroplastic isoform (GS 2) was present and active only in the leaves. The three GS isoforms were active in N-supplied (NH⁺ ₄ or NO ₃ ^– ) as well as in N-free-grown seedlings. This indicates (i) that a supply of nitrogen to the germinating seeds was not necessary for the induction of the GS isoforms and (ii) that no nitrogen-specific isoforms appeared during growth of seedlings with different nitrogen sources. The activity of GS, however, depended on the seedlings' nitrogen source: the specific activity was much higher in the leaves and much lower in the roots of NH⁺ ₄-grown barley than in the respective organs of NO ₃ ^– -fed or N free-grown plants. With increasing concentrations of NH⁺ ₄ (supplied hydroponically during growth), the specific activity of GS 1b increased in the leaves, but decreased in the roots. The activity of GS 2 (leaf) also increased with increasing NH⁺ ₄ supply, whereas GS 1a activity (leaf and root) was not affected. The changes in the activities of GS 1b and GS 2 were correlated with changes in the subunit compositions of the active holoenzymes: growth at increased levels of external NH⁺ ₄ resulted in an increased abundance of one of the four GS subunits, and of two of the five GS 1b subunits in the leaves. In the roots, however, the abundance of these two GS 1b subunits was decreased under the same growth conditions, indicating an organ-specific difference either in the expression of the genes coding for the respective GS 1b subunits or in the assembly of the GS 1b holoenzymes. Furthermore, growth at different levels of NH⁺ ₄ resulted in changes in the substrate affinities of the isoforms GS 1b (root and leaf) and GS 2 (leaf), presumably due to the changes in the subunit compositions of the active holoenzymes.Abbreviations FPLC fast protein liquid chromatography - GHA -glutamyl hydroxamate - GS glutamine synthetase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Role of the Plasmalemma in Controlling the Gravitropic Response of Wheat Coleoptiles

The changes in the specific binding of ³H-IAA to the plasmalemma from segments of wheat (Triticum aestivumL.) coleoptiles and the physiological activity of the IAA–protein complexes thus formed in dependence on the duration of gravitational stimulation (GS) (1 g) were studied. The overall inhibition of the formation of IAA–protein complexes was accompanied by a transverse polarization of their functional activity occurring as early as within two minutes after the onset of GS. The pretreatment of plasmalemmal vesicles with 0.1 M CaCl₂prevented the in vitroIAA–protein complex formation in the plasmalemma. It is suggested that the GS results in an increase in the plasmalemma permeability for Ca²⁺, which reduces the capacity of the plasmalemma to bind IAA at the early stages of the gravitropic response.     

Evidence for an ammonium transport system in free-living and symbiotic cyanobacteria

The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake. Initial accumulation (up to 60 s) was independent of CH₃NH ₃ ⁺ metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH₃NH ₃ ⁺ was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH₃NH ₃ ⁺ metabolism, but did not affect the CH₃NH ₃ ⁺ transport system.NH ₄ ⁺ , when added after the addition of ¹⁴CH₃NH ₃ ⁺ , caused the efflux of free CH₃NH ₃ ⁺ ; when added before ¹⁴CH₃NH ₃ ⁺ , NH ₄ ⁺ inhibited its uptake indicating that both NH ₄ ⁺ and CH₃NH ₃ ⁺ share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH₃NH ₃ ⁺ accumulation indicating that the transport system was -dependent. At pH 7 and at an external CH₃NH ₃ ⁺ concentration of 30 mol dm^-3, A. variabilis showed a 40-fold intracellular accumulation of CH₃NH ₃ ⁺ (internal concentration 1.4 mmol dm^-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a -dependent NH ₄ ⁺ transport system suggesting that the reduced inhibitory effect of NH ₄ ⁺ on nitrogenase cannot be attributed to the absence of an NH ₄ ⁺ transport system but is probably related to the reduced GS activity of the cyanobiont.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - pH transmembrane pH difference - TPMP⁺ triphenylmethylphosphonium     

Control of photosynthesis in barley mutants with reduced activities of glutamine synthetase and glutamate synthase

Heterozygous mutants of barley (Hordeum vulgare L. cv. Maris Mink) with decreased activities of chloroplastic glutamine synthetase (GS) between 97 and 47% of the wild type and ferredoxin dependent glutamate synthase (Fd-GOGAT) down to 64% of the wild type have been used to study aspects of glyoxylate metabolism and the effect of glyoxylate on the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo. In the leaf, the extractable activities of serine:glyoxylate aminotransferase decreased with a decrease in GS whereas activities of glutamate and alanine:glyoxylate aminotransferase increased, pointing to a re direction of amino donors from serine to glutamate and alanine. Under ambient conditions, the leaf contents of glutamate and alanine declined continuously with a decrease in GS, in parallel with the decrease in total amino acids. Glycine, serine and asparagine contents decreased with a decrease in GS to approximately 70% of the wild type, but increased again with a further decrease in GS. At high irradiances and at low CO₂ concentrations, glyoxylate contents exhibited a pronounced minimum between 60% and 80% GS. With a further decrease in GS, glyoxylate contents recovered and approached values similar to the wild type. The activation state of Rubisco showed a negative correlation with glyoxylate contents, indicating that a decrease in GS feeds back on the first step of carbon assimilation and photorespiration. The activation state of stromal fructose-1,6-bisphosphatase was unaffected by a decrease in GS or Fd-GOGAT, whereas the activation state of NADP dependent malate dehydrogenase changed in a complex manner. The CO₂photocompensation point, ^*, was appreciably increased in mutants with 47% GS. Mitochondrial respiration in the light (R_d) was reduced with a decrease in GS. Relative rates of CO₂ release into CO₂-free air between the wild type and the 47%-GS mutant correlated with determinations of ^*. These data are consistent with the view that when GS is decreased there is an increased oxidative decarboxylation of glyoxylate resulting from a decreased availability of amino donors for the transamination of glyoxylate to glycine, and that when GS activities are lower than 70% of the wild type an additional mechanism operates to reduce the photorespiratory loss of ammonia.Abbreviations AGAT nine:glyoxylate aminotransferase - FBPase fructose-1,6-bisphosphatase - Fd-GOGAT ferredoxin dependent glutamate synthase - GGAT glutamate:glyoxylate aminotransferase - GS glutamine synthetase - MDH malate dehydrogenase - PFD photon flux density - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SGAT serine:glyoxylate aminotransferase This research was supported by the Biotechnology and Biological Sciences Research Council initiative on the Biochemistry of Metabolic Regulation in Plants (PG 50/555).     

Bottom-up controls on a mixed-species HAB assemblage: A comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA

Recent novel mixed blooms of several species of toxic raphidophytes have caused fish kills and raised health concerns in the highly eutrophic Inland Bays of Delaware, USA. The factors that control their growth and dominance are not clear, including how these multi-species HAB events can persist without competitive exclusion occurring. We compared and contrasted the relative environmental niches of sympatric Chattonella subsalsa and Heterosigma akashiwo isolates from the bays using classic Monod-type experiments. C. subsalsa grew over a temperature range from 10 to 30 °C and a salinity range of 5–30 psu, with optimal growth occurring from 20 to 30 °C and 15 to 25 psu. H. akashiwo had similar upper temperature and salinity tolerances but also lower limits, with growth occurring from 4 to 30 °C and 5 to 30 psu and optimal growth between 16 and 30 °C and 10 and 30 psu. These culture results were confirmed by field observations of bloom occurrences in the Inland Bays. Maximum nutrient-saturated growth rates (μ_max) for C. subsalsa were 0.6 d⁻¹ and half-saturation concentrations for growth (K_s) were 9 μM for nitrate, 1.5 μM for ammonium, and 0.8 μM for phosphate. μ_max of H. akashiwo (0.7 d⁻¹) was slightly higher than C. subsalsa, but K_s values were nearly an order of magnitude lower at 0.3 μM for nitrate, 0.3 μM for ammonium, and 0.2 μM for phosphate. H. akashiwo is able to grow on urea but C. subsalsa cannot, while both can use glutamic acid. Cell yield experiments at environmentally relevant levels suggested an apparent preference by C. subsalsa for ammonium as a nitrogen source, while H. akashiwo produced more biomass on nitrate. Light intensity affected both species similarly, with the same growth responses for each over a range from 100 to 600 μmol photons m⁻² s⁻¹. Factors not examined here may allow C. subsalsa to persist during multi-species blooms in the bays, despite being competitively inferior to H. akashiwo under most conditions of nutrient availability, temperature, and salinity.