Factors regulating copper uptake in a cyanobacterium

Copper uptake in the diazotrophic cyanobacteriumNostoc calcicola was found to be typically biphasic, comprising rapid binding of the cations to the cell wall (during the first 10 min) followed by the subsequent metabolism-dependent intracellular uptake for at least 1 h, with a curvilinear kinetics saturating at 40 µM (K_m 25.0 µM, V_max 3.0 nmol Cu mg^–1 protein min^–1). The cellular Cu uptake was light- and ATP-dependent, and the addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or exogenous ATP proved that the energy to drive Cu transport was derived mainly through PS II reactions. The application of metabolic inhibitors and uncouplers like carbonylcyanidep-nitrofluoromethoxylphenyl hydrazone, N,N-dicyclohexycarbodiimide, azide, and p-chloromercuribenzoate revealed that -SH group(s), proton gradient across the cell membrane, and ATP hydrolysis were involved in the transmembrane movement of Cu inN. calcicola. While monothiol (2-mercaptoethanol) caused a twofold reduction in Cu uptake rate, dithiol (dithiothreitol) contributed towards a further drop in the cation uptake rate.     

Copper uptake by free and immobilized cyanobacterium

Abstract Copper uptake in free and immobilized cells of the cyanobacterium Nostoc calcicola has been examined. The immobilized cells invariably maintained a higher profile of Cu intake rate (12.7 nmol mg⁻¹ protein min⁻¹) over the free cells (6.0 nmol mg⁻¹ protein min⁻¹). The total Cu uptake in immobilized cells was almost two and a half-times more than their free cell counterpart under identical experimental conditions. Also, the immobilized cells showed a stronger positive correlation between Cu adsorption and uptake. The results have been discussed in terms of improved metabolic efficiency of immobilized cells.     

Methyl mercury uptake by free and immobilized cyanobacterium

Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein^–1 min^–1, 10 min) and total bioaccumulation (10.71 nmol mg protein^–1, 1 h) with a bioconcentration factor of 3.3×10³. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein^–1 min^–1, 10 min) with a saturation of 10.28 nmol mg protein^–1 reached within only 30 min (bioconcentration factor, 3.1×10³). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×10³). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein^–1 min^–1, 10 min), saturating at 30 min (8.81 nmol mg protein^–1), and the resultant lowest bioconcentration factor (2.7×10³). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells.     

Factors regulating cryIVB expression in the cyanobacterium Synechococcus PCC 7942

The expression of the larvicidal Bacillus thuringiensis subsp. israelensis cryIVB gene in cyanobacteria has been suggested to be an effective means of controlling mosquito populations. Using a variety of cryIVB constructs, in this study we have examined the effect of Synechococcus PCC 7942 culture age on intracellular toxin levels and have attempted to determine the mechanisms by which cryIVB gene expression is regulated. The data suggest that specific degradation of the cryIVB mRNA limits toxin production; however, the addition of cyanobacterial 3 untranslated DNA sequences to the cryIVB gene did not improve mRNA stability or toxin levels. An analysis of the cryIVB sequence and comparison of codon usage patterns with highly expressed cyanobacterial genes suggest that inefficient translation and intragenic ribosomal binding sites impede protein synthesis and result in rapid turnover of the toxin mRNA.     

Nickel uptake and its localization in a cyanobacterium

Abstract Nickel bioconcentration in different cell preparations of the cyanobacterium Nostoc muscorum was examined. A two- or three-fold increase in phosphate concentration over that prescribed in growth medium (58 μM), favoured nickel accumulation restricted to a threshold limit. Intact cells showed highest nickel bioconcentration (8.41 μmol mg⁻¹ dry wt) over spheroplasts (6.19 μmol mg⁻¹ dry wt) or polyphosphate bodies (5.88 μmol mg⁻¹ dry wt). Such preparations derived from similar cells indicate that the cyanobacterial cell wall could accommodate around 14–19% of the total nickel taken in by the cell with the overall nickel-bioconcentration sequence as: intact cells > spheroplasts > polyphosphate bodies > cell wall. The data suggest that polyphosphate bodies are the main sink for nickel.     

The ADP-ribosylation factor 1 (Arf1) is involved in regulating copper uptake

Copper is a cofactor for many essential enzymes in aerobic organisms. When intracellular copper levels are elevated, the Menkes (ATP7A) P-Type ATPase traffics from the trans-Golgi network (TGN) towards the plasma membrane to facilitate copper efflux. The ADP-ribosylation factor 1 (Arf1) is required for maintenance of Golgi architecture and for vesicular trafficking, including the copper-responsive trafficking of ATP7A. Here we report an ATP7A-independent role of Arf1 in copper homeostasis. Whilst the loss of ATP7A function increased copper levels, RNA interference mediated Arf1 knockdown reduced copper accumulation in HeLa cells as well as in both wild-type and ATP7A-null cultured fibroblasts. Arf1 therefore affected copper levels independently of ATP7A mediated copper efflux. Knockdown of Arf79F, the Drosophila melanogasterArf1 orthologue, also reduced copper accumulation in cultured Drosophila S2 cells, indicating an evolutionarily conserved role for this protein in cellular copper homeostasis. Whereas severe Arf1 inhibition with brefeldin A caused fragmentation and dispersal of the TGN resident protein Golgin 97, the peri-nuclear localisation of the Golgin 97 was retained following Arf1 knockdown, consistent with a moderate reduction in Arf1 activity. Ctr1 levels at the plasma membrane of cultured fibroblast cells were reduced following Arf1 knockdown, indicating an Arf1-dependent trafficking pathway is required for correct distribution of this copper uptake protein. Arf1-dependent trafficking pathways are therefore required for optimal copper uptake efficiency in cultured human and Drosophila cells.     

Factors regulating recruitment from the sediment to the water column in the bloom-forming cyanobacterium Gloeotrichia echinulata

1. The influence of light, temperature, sediment mixing and sediment origin (water depth) on the recruitment of the cyanobacterium Gloeotrichia echinulata was examined in the laboratory. 2. Light and temperature were the most important factors initiating germination in G. echinulata. 3. The extent of germination (recruited biovolume) was mainly regulated by temperature and sediment mixing. Furthermore, sediment mixing significantly enhanced the frequency of observed heterocysts and colonies. 4. Despite the fact that the deep and shallow sediments contained a similar number of akinete colonies, the highest recruitment occurred from shallow sediments, indicating that akinetes from shallow sediments have a higher viability than those from deeper parts of the lake. 5. Our results support the hypothesis that shallow sediments are more important than profundal sediments for the recruitment of G. echinulata to the pelagic zone.     

Hg2+ uptake in a cyanobacterium

The uptake of Hg²⁺ and its regulation in the cyanobacteriumNostoc calcicola Bréb. was studied. Hg²⁺ uptake pattern consisted of two distinct phases: (a) rapid binding of the cation to the negatively charged cell surface (first 10 min) and (b) its subsequent metabolism-dependent intracellular import, at least up to 40 min (saturating concentration 1.5 M Hg²⁺, K_m=1.0M Hg²⁺ and V_max 0.21 nmol Hg²⁺ mg^–1 protein min^–1). Hg²⁺ influx, to a major extent, depended on photosynthetically generated energy, and the supply of exogenous ATP (10 M) or DCMU (5 M) suggested the vital role of PS II-mediated energy to support the process. The significant lowering in Hg²⁺ uptake rate as well as total cellular Hg²⁺ in the presence ofp-chloromercuribenzoate (pCMB), azide (NaN₃), N,N-dicyclohexycarbodiimide (DCCD), and thiol (mercaptoethanol) indicated the role of membrane potential,-SH groups, and ATP hydrolysis in regulating Hg²⁺ transport. While Cu²⁺ antagonized Hg²⁺ intake, Ni²⁺ showed synergism.     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine     

Genes regulating copper metabolism

The metabolism of Cu is intimately linked with its nutrition. From gut to enzymes, Cu bioavailability to key enzymes and other components operates through a complex mechanism that uses transport proteins as well as small molecular weight ligands. Steps in Cu transport through the blood, absorption by cells, and incorporation into enzymes are slowly being understood. Cloning and sequencing of the genes for Menkes disease and Wilson disease has shown that membrane-bound enzymes analogous to Cu-ATPases in prokaryotes are equally important to Cu transport and homeostasis in mammalian cells. The primary structure of the mammalian Cu-ATPases has been deduced from cDNAs from tissues and organs. It now appears that mammalian Cu-ATPase have tissue and developmental specificity. In this review, we will focus on the Cu-ATPase that has been identified with Menkes disease. An emphasis will be placed on the existence of multiple forms of the ATPase and some indication as to how the different isoforms befit their role in the normal physiology of copper, specifically transmembrane transport and maintenance of a favorable internal cellular environment.     

Culture and nitrite uptake in immobilized Scenedesmus obliquus

Summary The green alga Scenedesmus obliquus was immobilized in Ca-alginate beads. The cell growth after immobilization was studied by cell counting. The nitrite uptake was not affected by immobilization, except that a longer lag phase was observed in immobilized cells than in free ones. That result could be due to a barrier effect of the matrix against nitrite diffusion inside the beads. The treatment of cells by glycerol prior to their immobilization in a batch reactor induced an increase of nitrite uptake by the cells. This effect disappeared after a few runs. The glycerol effect on specific rates seemed also to decrease when the number of immobilized cells increased. This decrease can be related to the decrease of light efficiency as well as substrate accessibility when a high cell concentration was used. Several alternating runs of Tris-HCl buffer containing nitrite growth medium depleted in combined nitrogen were tested. Cellular growth occurred inside the beads up to a maximum followed by a decrease of cell number in the beads.     

Nitrite uptake and its regulation in the cyanobacterium Anacystis nidulans

Two different components seem to participate in the uptake of nitrite by the cyanobacterium Anacystis nidulans, namely a transport system sensitive to N,N′-dicyclohexylcarbodiimide and a passive influx. The relative contribution of each component depended on the pH of the medium, that of the active system being prevalent at high pH values. The active transport of nitrite appears to be mediated by a high-affinity system, whereas the affinity for nitrite of the passive system is lower, similar to that of nitrite reductase. The utilization of nitrite was inhibited by products of the assimilation of ammonium via glutamine synthetase, apparently acting at the level of the active component involved in nitrite uptake.     

Cofactor recycling in an enzyme reactor. A comparison Using free and immobilized dehydrogenases with free and immobilized NAD

The requirements for enzymic cofactor recycling have been investigated in a system employing alcohol and lactate dehydrogenases. The interactions of various combinations of free dehydrogenases or dehydrogenases immobilized either to the same or separate supports, with free NAD, a soluble highmolecular weight derivative of NAD or an insoluble derivative of NAD have been examined.     

Continuous antibiotic production by an immobilized cyanobacterium in a seaweed-type bioreactor

The filamentous cyanobacterium,Scytonema sp. TISTR 8208, which produces a cyclic peptide antibiotic, was cultivated for 20 d in a seaweed-type bioreactor containing anchored polyurethan foam strips. Cells immobilized onto the foam strips produced the antibiotic for only several days, and the secreted antibiotic disappeared very rapidly from the medium. Cells accumulated the antibiotic intracellularly in a growth-related manner, and secreted it in the stationary phase. Since the antibiotic has a stable physico-chemical nature, the cells seem to take it up and metabolize it. When continuous cultivation was attempted, stable production of the antibiotic was maintained in the bioreactor for 16 d at a dilution rate of 0.01 h^–1. Three times more antibiotic was produced in the continuous culture than in the batch culture by the 16th day.     

Pathways of hydrogen uptake in the cyanobacterium Nostoc muscorum

Two pathways of hydrogen uptake in Nostoc muscorum are apparent using either oxygen or nitrogen as electron acceptor. Hydrogen uptake (under argon with some oxygen as electron acceptor assayed in the dark; oxyhydrogen reaction) is found to be more active in dense, light-limited cultures than in thin cultures when light is not limiting. Addition of bicarbonate inhibits this hydrogen uptake, because photosynthesis is stimulated. In a cell-free hydrogenase assay, a 10-fold increase of the activity can be measured, after the cells having been kept under lightlimiting conditions. After incubation under light-saturating conditions, no hydrogen uptake is found, when filaments are assayed under argon plus some oxygen. Assaying these cells under a nitrogen atmosphere, a strong hydrogen uptake occurs. The corresponding cell-free hydrogenase assay exhibits low hydrogenase activity. Furthermore, the hydrogen uptake by intact filaments under nitrogen in the light apparently is correlated with nitrogenase activity. These studies give evidence that, under certain physiological conditions, hydrogen uptake of heterocysts proceeds directly via nitrogenase, with no hydrogenase involved.Abbreviations Chl chlorophyll - DCMU (diuron) 3-3,4-dichlorophenyl)-1,1-dimethylurea - pev packed cell volume     

Hydrogen production by an immobilized cyanobacterium,Lyngbya sp.

The mesophilic, alkaliphilic, filamentous, and nonheterocystous fresh water cyanobacterium, Lyngbya sp. strain no. 108, was immobilized on calcium alginate gel. The optimum immobilizing conditions for hydrogen evolution were: 4% (w/v) alginate; 0.05 M CaCl₂; and 0.11 mg dry microbial cells/ml gel. The pH, temperature, and light intensity were 9.0, 30°C, and 1,000 1x, respectively. The optimum conditions for growth of immobilized cells were pH 9.5, 27°C, and 1,000 1x. Immobilized cells produced 25% more hydrogen than did free cells. The cells were incubated in a reaction mixture for hydrogen evolution and recultivated for 5 days in nitrogen-limited medium. These incubation and reactivation steps were repeated 3 times, after which, the cell yield and the amount of hydrogen evolution were 2.6- and 3.4-fold higher, respectively.     

ByDioscorea deltoidea free and immobilized plant cells

Summary 2-(4-methoxybenzyl)-1-cyclohexanone (1) was converted to its glucoside (4,5) via corresponding alcohols (3,4) usingDioscorea deltoidea Wall. free and immobilized plant cells.     

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.     

Hydrogen uptake by the nitrogen-starved cyanobacterium Anabaena cylindrica

The role of the oxyhydrogen reaction in the nitrogen metabolism of Anabaena cylin-drica, particularly under conditions of dinitrogen starvation, was investigated. It was shown that although this reaction supports nitrogenase activity in the dark, when the cells are deprived of nitrogen the rate of hydrogen uptake is little changed. Measurements of ammonia excretion into the medium in the presence of methionine sulfoximine under such conditions indicated that hydrogen uptake supported the turnover of cell protein as an alternative source of nitrogen. In the absence of H₂ and O₂ in the dark, nitrogenase activity was negligible but protein turnover continued. In their presence nitrogenase activity was greatly stimulated; turnover was also stimulated but to a greater extent in the absence of nitrogenase substrates. The oxyhydrogen reaction also stimulated uptake of ammonium ions by intact filaments in argon in the dark. Only at very low hydrogen tensions can net hydrogen formation be obtained in argon/CO₂ in the light, casting considerable doubt on the suitability of hydrogenase-containing organisms for biophotolytic hydrogen formation. Addition of exogenous ammonia to the cultures incubated in argon resulted in a pronounced stimulation of H₂ uptake; nitrate and its derivatives had no such effect, nor did various amino acid derivatives of ammonia.