Hexose-transport-deficient mutants of Chlorella vulgaris

Autotrophically grown cells of Chlorella vulgaris show a strong increase in the uptake rates for hexoses and for seven amino acids when incubated in the presence of hexoses. This increase is due to de-novo synthesis of three transport proteins: one forhexoses and two for amino acids. Mutants deficient in hexose transport were obtained after treatment of wild-type cells with acridine orange, followed by a selection procedure using the toxic hexose analogue, 2-deoxy-D-glucose. Moreover, the two amino-acid-transport systems could not be induced in these mutants by hexoses. The capacity to phosphorylate hexoses was identical in mutants and in the wild-type strain. The loss of transport activities can be correlated with the loss of certain radiolabeled protein bands on fluorograms of sodium dodecylsulfate-polyacrylamide gels. These proteins are assumed to be responsible for the different transport systems in the wild-type strain. With the help of additional mutants defective in one or two of the different aminoacid-transport systems, it has been attempted to assign the different transport activities to individual protein bands on the gel.Abbreviations AUP arginine-uptake-defective mutant - 2-DG 2-deoxy-D-glucose - 6-DG 6-deoxy-D-glucose - HUP hexose-uptake-defective mutant - PUP^- proline-uptake-defective mutant - SDS sodium dodecyl sulfate - WT wild type     

Spectrophotometry of individual Chlorella cells]

A research of optical properties of individual microalgae Chlorella vulgaris cells was performed under various conditions of cultivation. On the basis of cell absorption spectra measurements in the visible spectrum region chlorophyll "a" absorption maxima are estimated to fall within the intervals of 669-675 nm for the red region of the spectrum and 429--436 nm for the blue one. Concentration of chlorophylls in the cells of the diameter from 2,8 up to 3,3 micrometers was found to increase considerably for algae grown both in continuous denisty static and periodical regimes. Integral optical density of individual Chlorella cells in the range of 400-750 nm grows with cell diameter increase. This increase appears to be most intensive in the cells grown in density static regime. A correlation was also established between algae cell dimensions and the general maxima position (correlation coefficient has a positive sign and absolute value near unit). With cell diameter growth the absorption maxima shift to the region of long waves.     

Blue-light-control of the uptake of amino acids and of ammonia in Chlorella mutants

In non-photosynthetic, yellow or colourless mutant cells of Chlorella kessleri , grown with nitrate as sole nitrogen source, blue light inhibited the uptake of the amino acids glycine, proline and arginine and of ammonia in growing cells, while it enhanced the uptake of these amino acids in resting cells. On the other hand, in cells grown with ammonia as the only nitrogen source without nitrate reductase activity, blue light did not influence the uptake of amino acids and of ammonia in growing cells, while it enhanced the uptake of amino acids in resting cells. Addition of methionine sulphoximine, a potent inhibitor of glutamine synthetase, to growing cells, resulted in intracellular ammonia-accumulation and inhibition of uptake of glycine and of ammonia. For the colourless mutant, blue light was shown to activate purified nitrate reductase. These results indicate that in the mutant cells of Chlorella examined, uptake of ammonia seems to be influenced by nitrate reductase and the uptake of amino acids was influenced by both nitrate reductase and an unknown blue-light-receptor(s). The uptake of urea in mutant cells is not influenced by the irradiation with blue light. Uptake of glycine was also increased after addition of glucose (hexose) in the dark. Because blue light is known to enhance the breakdown of starch, a reaction producing glucose for oxidative degradation in the algae used, the role of glucose (hexose) in the blue light-affected uptake of amino acids is discussed.     

The concentration of Cytochrome f and P700 in chlorophyll-deficient mutants of Chlorella fusca

The ratio of Chlorophyll: Cytochrome f and of Chlorophyll: P700 (reaction center pigment in photosystem I) is essentially lower in chlorophyll-deficient mutants than in the normal green strain. On a dry weight basis, the mutants have the same or a higher content of redox enzymes than the normal form. The size of the photosynthetic unit of the mutants is 4 to 7 times smaller than that of the normal strains, due mainly to a deficiency of the light-harvesting chlorophyll-protein complex.Abbreviations Chl chlorophyll - Cyt f Cytochrome f - P700 reaction center pigment in photosystem I - PS photosystem - LH light-harvesting     

Irreversible inactivation of Chlorella glutamine synthetase by urea]

The effect of urea on Chlorella glutamine synthetase (E. C. 6.3.1.2) activity and tertiary structure is investigated. Urea is found to inhibit the activity of glutamine synthetase, the inhibitory effect being independent on the time. The enzyme molecule relax and changes its affinity to ammonium under the effect of urea at concentrations of 1.0-4.0 M. Higher concentrations of urea (5,0 M and more) produce a dissociation of the enzyme molecule into monomers without any intermediate forms. Monomers do not possess any synthetase and transferase activities. Substrates and cofactors do not protect the enzyme from the effect of urea and do not stimulate the emzyme reactivation and reaggregation after its dissotiation. The data obtained are discussed from the viewpoint of the regulation of Chlorella glutamine synthetase activity in vivo.     

NMR-relaxation in hydrated collagen from the spotted dogfish]

Collagen samples from dog-fish egg case at different water content were studied by the 1H NMR relaxation method. The dependences of the proton spin-lattice and spin-spin relaxation rates on the concentration of water in hydrated native collagen were measured. The fractions of water protons of different mobility and their corresponding spin-spin and spin-lattice relaxation rates were determined in a multi-phase model of water protons in natural biopolymer-water systems. The correlation times were calculated as the characteristics of molecular motion in hydrated collagens with different content of absorbed water. The results obtained were compared with literature data of pulse NMR studies of molecular mobility in other collagen fibers.     

Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids]

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.     

Cytochrome f from Chlorella pyrenoidosa Pringsheim 82 T]

Cytochrome of the f type was isolated from the thermophilous autotrophic strain Chlorella pyrenoidosa Pringsheim 82T and purified on Sephadex G-75. The isolation procedure allowed a simultaneous production of glutamate dehydrogenase isoenzymes. From 100 g of Chlorella wet paste 100 to 120 nM of electrophoretically unicomponent protein with a molecular weight of 12,000 to 13,000 were isolated. The Chlorella cytochrome had an absorption spectrum in the visible light that was typical of the f type cytochromes.     

Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k)

 A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO₃ ⁻ metabolism. Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones. The final screening was performed using NO₃ ⁻, NO₂ ⁻ or NH⁺ ₄ as N-source. The mutants isolated absorb NO₃ ⁻ with rates close to those measured for the wild type and they excrete NO₂ ⁻ into the medium. The ratio between NO₃ ⁻ uptake and NO₂ ⁻ excretion was 1:1. The sensitivity of NO₃ ⁻ uptake to NH⁺ ₄ was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of the wild-type) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling of C and N metabolism by inorganic N-compounds. Received: 8 October 1999 / Accepted: 25 January 2000     

Production of auxotrophic mutants of Shigella sonnei]

Use of selection method with R. Ffm phage offered a possibility of obtaining R-forms of Sh. sonnei from the population of bacteria of phase II with a low frequency of natural dissociation. Mono- and polyauxotrophic mutant of I, II and R-phases by hir, ilv, pro, and mtl markers were obtained by treatment of Sh. sonnei strains with nitrosoguanidine and ethyl-methanesulfonate. The frequency of mutation in two Sh. sonnei strains studied in this work (UBC and No. 941) by the same genetic regions proved to differ. The change of Sh. sonnei phase I into phase II was observed only in selection by proline marker which could be connected with the existence in this region of a gene controlling the dissociation process.     

Spotlight on spotted mice: a review of white spotting mouse mutants and associated human pigmentation disorders

Mutation of genes that regulate neural crest-derived melanoblast development and survival can result in reduction and/or loss of mature melanocytes. The reduction in melanocyte number in the skin and hair follicles manifests itself as areas of hypopigmentation, commonly described as white spotting in mice. To date ten genes have been identified which are associated with white-spotting phenotypes in mouse. Seven of these genes are associated with neural crest and melanocyte disorders in humans. This review summarizes the phenotypes associated with mutation of these genes in both mouse and man. We describe our current understanding of how these genes function in development, and explore their complex roles regulating the various stages of melanocyte development.     

Purification, properties and quaternary structure of glutamine synthetase from Chlorella]

A highly purified preparation of glutamine synthetase from chlorella grown on a medium containing nitrate as a sole source of nitrogen, was isolated and characterized by disc-electrophoresis and analytical ultracentrifugation. The N-terminal amino acid of glutamine synthetase is glycine. The molecular weight of glutamine synthetase is 32.000; its activity in the presence of Mg2+ was 150 mkmol o-phosphate per min per mg protein. The molecular weight of subunits of the enzyme, equal to 53.000 was determined by disc-electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. Electron microscopy of negatively contrasted enzyme preparations revealed 6 subunits in the enzyme molecule, arranged in a point symmetry group 32.     

Bioconversion of steroids in mutants of Nocardia restrictus]

Nocardia restrictus grows quickly on synthetic media containing different carbonated substrates (steroids, organic acids). The control of growth parameters of this microbial species allowed the development of the production and selection methodology for mutants unable to grow on androst-4ène-3,17-dione. A stable mutant convert androst-4-ène-3,17-dione in perhydroindan propionic acid without addition of any degradation inhibitor in the medium.     

Enhanced production of triacylglycerols and polyunsaturated fatty acids in novel acid-tolerant mutants of the green microalga Chlorella saccharophila

Bioprocess and Biosystems Engineering - In this study, the microalga Chlorella saccharophila was subjected to ultraviolet (UV) mutagenesis, and mutant screening was conducted based on acidity...     

Eremothecium ashbyii mutants resistant to 2,6-diaminopurine]

3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.