Regulation of urea uptake by ammonia in the cyanobacterium Anabaena doliolum

Abstract The urea uptake system was studied with regard to its repression and derepression in the cyanobacterium, Anabaena doliolum . The uptake of urea was energy-dependent and was repressed in ammonia grown cells. Repression of the urea uptake by ammonium did not require ammonium assimilation or de novo protein synthesis, suggesting that ammonium itself was the repressor signal. The derepression of the urea uptake system, however, required de novo protein synthesis and glutamine synthetase activity.     

Nickel is a component of hydrogenase in Rhizobium japonicum

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.     

Xylose and some non-sugar carbon sources cause catabolite repression in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Glucose and other sugars, such as galactose or maltose, are able to cause carbon catabolite repression in Saccharomyces cerevisiae. Although glycolytic intermediates have been suggested as signal for repression, no evidence for such a control mechanism is available. The establishment of a correlation between levels of intracellular metabolites and the extent of catabolite repression may facilitate the identification of potential signal molecules in the process. To set a framework for such a study, the repression produced by xylose, glycerol and dihydroxyacetone upon genes belonging to different repressible circuits was tested, using an engineered strain of S. cerevisiae able to metabolize xylose. Xylose decreased the derepression of various enzymes in the presence of ethanol by at least 10-fold; the corresponding mRNAs were not detected in these conditions. Xylose also impaired the derepression of galactokinase and invertase. Glycerol and dihydroxyacetone decreased 2- to 3-fold the derepression observed in ethanol or galactose but did not affect invertase derepression. For yeast cells grown in media with different carbon sources, no correlation was found between repression of fructose-1,6-bisphosphatase and intracellular levels of glucose 6-phosphate or fructose 1,6-bisphosphate.     

Aspects of inorganic nitrogen assimilation in yeasts

Cultures of Candida utilis utilise glutamate in preference to ammonia and ammonia in preference to nitrate. The nitrate reductase of this organism is induced by nitrate and repressed in cultures grown on glutamate or ammonia. Nitrate-grown cultures of C. utilis, irrespective of the medium nitrate concentration, behave as though nitrogen-limited. In contrast to C. utilis, Saccharomyces cerevisiae utilises ammonia in preference to glutamate. In eight yeasts studied the highest cellular contents of biosynthetic NADP-linked glutamate dehydrogenase were found in batch cultures containing low concentrations of ammonia or in nitrogen-limited chemostat cultures. NAD-linked glutamate dehydrogenase activity was detected in extracts of cells grown in the presence of glutamate but not in those grown in the presence of ammonia.     

Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120

Two distinct types of hydrogenase occur in Anabaena 7120 and are distinguishable in whole filaments by the application of selective assay methods. A reversible hydrogenase occurs both in heterocysts and vegetative cells and can be selectively assayed by measuring H2 evolution from reduced methyl viologen. Activities in aerobically grown filaments were low but could be increased by 2 to 3 orders of magnitude by growing cells microaerobically. The presence of the reversible hydrogenase was independent of the N2-fixing properties of the organism, and activity did not respond to added H2 in the culture. Illumination was necessary during derepression of the reversible hydrogenase, and addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea increased the amount of enzyme that was synthesized. An uptake hydrogenase occurred only in heterocysts of aerobically grown filaments, but a small amount of activity also was present in the vegetative cells of filaments grown microaerobically with 20% H2. It was assayed selectively by measuring an oxyhydrogen reaction at atmospheric levels of O2. Additional uptake hydrogenase could be elicited by including H2 or by removing O2 from the sparging gas of a culture.     

Effect of unsaturated fatty acids on the biosynthesis of glucose-repressed enzymes in yeast

In anaerobically glucose-grown yeast isocitrate lyase (EC 4.1.3.1.), malate synthase (EC 4.1.3.2.) and malate dehydrogenase (EC 1.1.1.37.) are repressed by glucose. 24 h cultures still contain 0.3–0.4% glucose in the medium, which is enough to completely repress these activities. Aeration of these cells, in buffer containing acetate, initiates the formation of the three enzymes. Within 16 h, the specific activities of these enzymes increase about 140, 120 and 70-fold, respectively. Glucose-6-phosphate dehydrogenase activity was not altered. When the yeast was grown anaerobically, but with a supplement of an unsaturated fatty acid in the medium, synthesis of the three enzymes was much faster and the specific activities after 16 h of derepression were considerably higher. A relationship exists between the number of double bonds in the unsaturated fatty acid molecule and its capability to stimulate enzyme synthesis: linolenic acid is more effective than linoleic acid, which, in turn, is much more effective than oleic acid. Increasing periods of aeration with glucose of anaerobically grown cells prior to derepression results in an increasing stimulation of enzyme synthesis on subsequent derepression. Anaerobic incubation of yeast in the presence of an unsaturated fatty acid in advance to derepression also increased the velocity of enzyme formation. It is suggested that during the aeration period with glucose and during anaerobic incubation with an unsaturated fatty acid a more active protein synthesizing apparatus was formed.     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

Interaction between ribulose 1,5-bisphosphate carboxylase/oxygenase activity and the ammonia assimilatory system of Rhodobacter sphaeroides.

The levels of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides were found to depend on the concentration of ammonia supplied to photolithoautotrophically grown cultures. Under conditions in which the cells rapidly depleted the available ammonia, the level of in situ RubisCO activity decreased to less than 5% maximum activity; even at its maximum level under these conditions, the RubisCO activity was only 5% of the activity obtained from cultures supplied with saturating levels of ammonia. When cells were incubated with somewhat higher but not saturating amounts of ammonia, in situ RubisCO activity decreased immediately after the cells depleted the cultures of ammonia. The decrease in activity was not due to any detectable degradation of RubisCO protein, indicative of some mechanism to regulate the activity of the enzyme in response to the intracellular levels of assimilated ammonia. Furthermore, under conditions optimum for RubisCO inactivation, in situ RubisCO activity in permeabilized whole cells greatly exceeded the levels of enzymatic activity determined in vitro in cell extracts. Blockage of ammonia assimilation by inhibition of glutamine synthetase with methionine sulfoximine prevented the recovery of form I RubisCO from pyruvate-mediated inactivation, suggesting the presence of regulatory mechanisms common to both CO2 fixation and ammonia assimilation.     

Changes in nuclear proteins of astrocytes as a result of acute ammonia or ethanol exposure

We have used an in vitro system to monitor the effects of high levels of ammonia and ethanol on glial cells. Nuclei were isolated and the protein profiles examined by two-dimensional gel electrophoresis. Acute exposure of rat astrocyte cell cultures to ammonia or ethanol resulted in changes in cellular morphology and the level of some nuclear proteins. Glial fibrillary acidic protein (GFAP) levels remained constant under both treatments. Several nuclear proteins were increased specifically. Only one protein was visually detected which was unique to treatment with ammonia or ethanol. This protein (p2a) appeared only in the presence of ammonia. There were no changes in previously observed astrocyte-associated proteins (Silverman et al. Neurochem Int. 12, 513–518, 1988). Two proteins appeared de novo upon either treatment with either ammonia or ethanol. These latter proteins had a molecular weight and pI profile similar to the major class of nuclear stress proteins (hsp70). However, results from immunoblot experiments clearly demonstrated that hsp70 was not induced in astroycte cultures following exposure to ammonia or ethanol.     

Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h^–1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.     

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Summary Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR).The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis.One class of mutants lacked NR activity (nar ^-) whereas the specific activity of NR was low in another class of mutants (nar ^def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.     

Regulation of beta-1, 4-Glucosidase Expression by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 expressed beta-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed beta-glucosidase expression (<0.3 U/ml); however, this yeast did produce beta-glucosidase when the initial glucose concentration was 相似文献   

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

Glutamine synthetase activity in the ruminal bacterium Succinivibrio dextrinosolvens.

Succinivibrio dextrinosolvens C18 was found to possess glutamine synthetase (GS), urease, glutamate dehydrogenase, and several other nitrogen assimilation enzymes. When grown in continuous culture under ammonia limitation, both GS and urease activities were high and glutamate dehydrogenase activity was low, but the opposite activity pattern was observed for growth in the presence of ample ammonia. The addition of high-level (15 mM) ammonium chloride to ammonia-limited cultures resulted in a rapid loss of GS activity as measured by either the gamma-glutamyl transferase or forward assay method with cells or extracts. No similar activity losses occurred for urease, glutamate dehydrogenase, or pyruvate kinase. The GS activity loss was not prevented by the addition of chloramphenicol and rifampin. The GS activity could be recovered by washing or incubating cells in buffer or by the addition of snake venom phosphodiesterase to cell extracts. Manganese inhibited the GS activity (forward assay) of untreated cells but stimulated the GS activity in ammonia-treated cells. Alanine, glycine, and possibly serine were inhibitory to GS activity. Optimal pH values for GS activity were 7.3 and 7.4 for the forward and gamma-glutamyl transferase assays, respectively. The glutamate dehydrogenase activity was NADPH linked and optimal in the presence of KCl. The data are consistent with an adenylylation-deadenylylation control mechanism for GS activity in S. dextrinosolvens, and the GS pathway is a major route for ammonia assimilation under low environmental ammonia levels. The rapid regulation of the ATP-requiring GS activity may be of ecological importance to this strictly anaerobic ruminal bacterium.     

Glutamine synthetase activity in the ruminal bacterium Succinivibrio dextrinosolvens

Succinivibrio dextrinosolvens C18 was found to possess glutamine synthetase (GS), urease, glutamate dehydrogenase, and several other nitrogen assimilation enzymes. When grown in continuous culture under ammonia limitation, both GS and urease activities were high and glutamate dehydrogenase activity was low, but the opposite activity pattern was observed for growth in the presence of ample ammonia. The addition of high-level (15 mM) ammonium chloride to ammonia-limited cultures resulted in a rapid loss of GS activity as measured by either the gamma-glutamyl transferase or forward assay method with cells or extracts. No similar activity losses occurred for urease, glutamate dehydrogenase, or pyruvate kinase. The GS activity loss was not prevented by the addition of chloramphenicol and rifampin. The GS activity could be recovered by washing or incubating cells in buffer or by the addition of snake venom phosphodiesterase to cell extracts. Manganese inhibited the GS activity (forward assay) of untreated cells but stimulated the GS activity in ammonia-treated cells. Alanine, glycine, and possibly serine were inhibitory to GS activity. Optimal pH values for GS activity were 7.3 and 7.4 for the forward and gamma-glutamyl transferase assays, respectively. The glutamate dehydrogenase activity was NADPH linked and optimal in the presence of KCl. The data are consistent with an adenylylation-deadenylylation control mechanism for GS activity in S. dextrinosolvens, and the GS pathway is a major route for ammonia assimilation under low environmental ammonia levels. The rapid regulation of the ATP-requiring GS activity may be of ecological importance to this strictly anaerobic ruminal bacterium.     

Chronic ethanol increases liver plasma membrane fluidity

Purified plasma membrane fractions of cultured well-differentiated Reuber H35 hepatoma cells were studied after growth in the presence or absence of ethanol. Growth of cells in the presence of ethanol significantly increased plasma membrane 5'-nucleotidase activity but did not influence sodium-potassium adenosinetriphosphatase activity. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from cells grown in 80 mM ethanol for 3 weeks, compared to controls. Decreased polarization of DPH in plasma membranes was observed after 3-weeks growth of cells in as little as 1 mM ethanol. A 1-h exposure to 80 mM ethanol had no effect. Altered DPH polarization was due to a decrease in the order parameter of the probe. The rotational correlation time of the probe was virtually unchanged. Chronic ethanol treatment of cells did not alter the polarization of the membrane surface probe trimethylammoniodiphenylhexatriene. Plasma membranes from cells grown in 80 mM ethanol had decreased contents of both phospholipid and unesterified cholesterol, but the cholesterol to phospholipid ratio was unchanged. The percentages of sphingomyelin and phosphatidylserine in plasma membrane phospholipids were significantly decreased after ethanol treatment, while the phosphatidylcholine/sphingomyelin ratio was increased by 42%. Vesicles prepared from total plasma membrane lipids of ethanol-treated cells, as well as vesicles prepared from polar lipids alone, showed the same alterations in DPH polarization as did plasma membranes. The importance of ethanol metabolism in the observed plasma membrane changes was demonstrated in two ways.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum.

Rhizobitoxine produced by Bradyrhizobium species strongly prevented derepression of hydrogenase expression in free-living Bradyrhizobium japonicum, although the toxin had no effect on the activity of cells which had already synthesized hydrogenase protein. Dihydrorhizobitoxine, a structural analog of rhizobitoxine, proved to be a less potent inhibitor of hydrogenase derepression. Rhizobitoxine did not cause cell death at a concentration sufficient to eliminate hydrogenase expression. The large subunit of hydrogenase was not detectable with antibody after derepression in the presence of rhizobitoxine. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different in the presence or absence of rhizobitoxine. These results indicated that rhizobitoxine inhibited hydrogenase synthesis in free-living B. japonicum. Cystathionine and methionine strongly prevented the inhibition of hydrogenase derepression by rhizobitoxine, suggesting that the inhibition involves the level of sulfur-containing amino acids in the cell.     

Regulation of glutamine uptake in the cyanobiont Nostoc ANTH

Abstract Glutamine uptake in the cyanobiont Nostoc ANTH was energy-dependent and repressed in ammonia-grown cells. l -Methionine- dl -sulphoximine (MSX), a glutamate analogue and an inhibitor of glutamine synthetase (GS), did not affect glutamine uptake whereas azaserine, an inhibitor of glutamate synthase (GOGAT) did, suggesting that GS activity is not necessarily involved in the glutamine uptake system and that increased intracellular glutamine level regulates its own uptake. Repression of glutamine uptake by ammonia did not require de novo protein synthesis but required GS activity, suggesting that ammonia itself was not the repressor signal. The derepression of the glutamine uptake system did not require GS activity but required de novo protein synthesis.