Roy Foster

Osmotic adjustment in cyanobacteria from hypersaline environments

The intracellular concentrations of the monovalent inorganic cations K+ and Na+, low molecular weight carbohydrates and quaternary ammonium compounds have been determined for 4 strains of cyanobacteria (Aphanothece halophytica, Coccochloris elabens, Dactylococcopsis salina and Synechocystis DUN52) originally isolated from hypersaline habitats (i.e. habitats with a salinity greater than that of seawater) over a range of external salt concentration (from 50% to 400% seawater). Intracellular cation levels (Na+ and K+) were determined to be within the range 80–320 mmol · dm-3 (cell volume), showing only minor changes in response to salinity. Intracellular carbohydrates were found to comprise a negligible component of the intracellular osmotic potential [at 2–19 mmol · dm-3 (cell volume)], throughout the salinity range. Quaternary ammonium compounds, however, were recorded in osmotically significant quantities [up to 1,640 mmol · dm-3 (cell volume)] in these strains, showing major variation in response to salinity. Thus Synechocystis DUN 52 showed an increase in quaternary ammonium compounds in the oder of 1,200 mmol · dm-3 between 50% and 400% seawater medium, accounting for a significant proportion of the change in external osmotic potential.Examination of intact cells and cell extracts using 13C and 1H nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of the quaternary ammonium compound glycine betaine as the major osmoticum in the 4 strains; no other compounds were detected during NMR assays. These results suggest a common mechanism of osmotic adjustment, involving quaternary ammonium compounds, in cyanobacteria from hypersaline environments.

Osmotic adjustment in Spirulina platensis

The filamentous cyanobacterium Spirulina platensis has been examined for salt tolerance and osmotic adjustment. Salinities up to 150% seawater had little effect on growth yield or photosynthetic O2 evolution; higher salinities were markedly inhibitory. Osmotic adjustment was achieved by the intracellular accumulation of the low-molecular-weight carbohydrate glucosyl-glycerol in response to increased external salinity: in fullstrength (100%) seawater glucosyl-glycerol accounted for approximately 5.0% of the dry weight of the cyanobacterium. Trehalose was also present, particularly in cells at low salt concentration, and in 50% seawater medium accounted for up to 1.0% of the dry weight of the cyanobacterium. For cells grown in 100% seawater the ratio of trehalose to glucosyl-glycerol varied with temperature: at 37°C trehalose comprised 31% (w/w) of the low-molecular-weight carbohydrates while at 20°C only 9% of the total was trehalose. When subjected to hypo-osmotic shock the intracellular concentration of glucosyl-glycerol decreased and this was mirrored by an increase in glycogen. An understanding of the osmotic adjustment of S. platensis has implications both for the mass culturing of this and other strains of Spirulina and possibly also for the quality of the harvested product.

Organic solute accumulation in osmotically-stressed Enteromorpha intestinalis

The role of organic solutes in the osmotic adjustment processes of the marine macroalga Enteromorpha intestinalis (L.) Link was investigated in 1986, using fresh samples collected from mid-shore rock pools at Tayport, Fife, Scotland. Natural-abundance 13C nuclear magnetic-resonance spectroscopy revealed β-dimethylsulphoniopropionate (DMSP) to be the only major low molecular weight organic osmolyte present. However, on transfer to a hypersaline medium (300% sea water; 100%=35 S‰), tissue sucrose and proline levels increased markedly, while DMSP remained constant. Recovery of optimal photosynthetic activity and increases in inorganic ion levels occurred over a similar time scale to the changes in sucrose and proline (within 48 h), indicating that these two organic solutes are involved in hyperosmotic adjustment in E. intestinalis while DMSP is not. Freshly-collected plants transferred to 300% sea water medium in the dark showed no significant increases in organic osmolytes. In contrast, starch-enrichment (16 d continuous illumination) led to enhanced synthesis of sucrose and proline in the light and in darkness, but tissue DMSP levels showed no variation throughout. These observations suggest that DMSP is not involved in short-term osmoacclimation in E. intestinalis.

Identification and quantification of Methylated Osmolytes in algae using Proton Nuclear Magnetic Resonance Spectroscopy

A method is described for the rapid determination of osmotically significant amounts of the methylated solutes glycine betaine and β-dimethylsulphoniopropionate (DMSP) in algal extracts, using 1H nuclear magnetic resonance techniques. The method is straightforward, since algal extracts require no further purification prior to analysis. The procedure is sensitive, non-destructive and may also be applied to other quaternary ammonium and tertiary sulphonium compounds.

Complexes of pyrene with 2,4,6-trinitroanisole. Studies of association in solution and the crystal structure of the 1:1 complex

Abstract Cyclohexane solutions of 2,4,6-trinitroanisole (A) containing excess pyrene (D) can be described in terms of a double equilibrium involving complexes DA and D2A. Formation constants for these complexes from A and from DA respectively from three independent experiments are in good agreement, the average values being K1= 9.7 kg mol-1 and K2= 1.8 kg mol-1 at 33.5°. The crystalline complex was obtained by gel diffusion. The mw (C23H15N3O7) corresponds to 1:1 stoichiometry C16H10, C7H5N3O7, X-ray structure data: P21/c, a = 10.633(7), b= 16.336(8), c = 11.683(7) A, β = 94.62(12)°, V = 2023 A3, F000 = 924, μ(CuKα) = 8.3 cm-1, Z = 4. R = 0.091 for 1572 reflexions. The crystal contains extended stacks ADADA, parallel to (101). The pyrene molecules are disordered by rotation over two sites. The majority site has an occupancy factor of 0.544(2). The angle between the normals to the mean planes of A and D is 7.0(4)°, allowing the nitro groups of A to twist out of the plane of the benzene ring by 41.0(1.0), 2.7(5) and 20.7(8)°.

13 C nuclear magnetic resonance studies of the products of reaction of acetaldehyde and of simple ketones in liquid ammonia, in hydrazine hydrate, and in some substituted hydrazine solutions

13 C N.m.r. shows that acetaldehyde rapidly forms the classical ‘aldehyde–ammonia’ in liquid ammonia, together with 2,4,6-trimethylhexahydro-1,3,5-triazine. For the first time, the formation of gem-aminohydroxy products corresponding to aldehyde–ammonia adducts by a range of ketones under the same conditions has been observed directly. In parallel studies, the products formed by acetaldehyde and by various ketones in hydrazine hydrate and in some simply substituted hydrazines have also been determined. It has been possible not only to detect hydrazones, azines, and a hexahydrotetrazine with the appropriate reagents, but also to identify the formation of gem-hydrazino-hydroxy adducts corresponding to aldehyde–ammonia adducts.

Altritol in the brown alga Himanthalia elongata

Abstract Natural abundance 13 C NMR spectroscopy has shown that altritol (talitol) is a major constituent within vegetative and reproductive tissues of Himanthalia elongata . Quantitative GC studies have confirmed that altritol concentrations are 80–160% higher than for the hexitol mannitol (which occurs throughout the Phaeophyta). The data suggest an osmotic role for altritol.

The reactions of two σ-anionic compounds (Jackson–Meisenheimer adducts) in liquid ammonia studied by 13C nuclear magnetic resonance spectroscopy

The reactions of the Jackson–Meisenheimer adducts derived from: (a) methoxide ion and 1,3,5-trinitrobenzene; (b) acetone carbanion and 1,3,5-trinitrobenzene, in liquid ammonia solution have been studied by 13C n.m.r. With the adduct (a) the methoxy group is labile as expected, whereas with (b) the C–C bond on the ring sp3 carbon atom remains unbroken, but cyclisation through the γ-carbon of the acetonyl group occurs to form a bicyclo-derivative.

13 C nuclear magnetic resonance of Jackson–Meisenheimer (σ-anionic)-type products formed when 1,3,5-trinitrobenzene, and when 2,4,6-trinitrotoluene, are dissolved in liquid ammonia

1 : 1 and 2 : 1 products formed by nucleophilic attack of ammonia in liquid ammonia solutions of 1,3,5-trinitrobenzene and of 2,4,6-trinitrotoluene have been observed by 13C n.m.r. spectroscopy. In the latter reaction three 2 : 1 products are observed: two geometrical isomers formed by attack at the 1- and 3-position of 2,4,6-trinitrotoluene and a structural isomer formed by attack at the 3- and 5-position.

Use of internal and external references in nuclear magnetic resonance determinations of association constants of weak molecular complexes

For each of three typical weak electron-donor–acceptor interactions in dilute solution in an “inert” solvent, the values of association constants, based on n.m.r. chemical shift measurements, are the same whether an internal or an external reference is used.