Role of relA mutation in the survival of amino acid-starved Escherichia coli

Amino acid-starved cells of Escherichia coli relA ⁺, which contain a large number of glycogen particles, are able to survive in phosphate buffer for a longer time period than their relaxed counterparts. With regard to NH ₄ ⁺ starvation differences in the survival of both strains were not found. NH ₄ ⁺ starved cells of E. coli relA are able to synthesize glycogen but amino acid-starved cells of the relA strain are not. We suggest that the synthesis of glycogen triggered by guanosine tetraphosphate during amino acid starvation is responsible for the prolonged viability of the E. coli relA ⁺ strain.Abbreviations ppGpp guanosine tetraphosphate     

Levels of trehalose and glycogen inArthrobacter globiformis under conditions of nutrient starvation and osmotic stress

Cells ofArthrobacter globiformis grown in carbohydrate-rich media were found to contain large quantities of low-M_r carbohydrates (800 g/mg protein) and only small amounts of amino acids, in addition to high amounts of glycogen (2 mg/mg protein). At increasing osmotic values of the medium, low-M_r carbohydrate levels increased to 1300 g/mg protein. Low-M_r pools were extracted from the cells with hot 75% ethanol, and subjected to thin layer, gel and gas-liquid chromatography. They turned out to consist mainly of ,-trehalose. Levels of trehalose inArthrobacter cells have the tendency to remain constant, both during nutrient exhaustion (resulting in glycogen consumption), and on addition of excess of carbon source to the medium (resulting in an increased glycogen content of the cells). The stress-tolerant properties ofArthrobacter (resistance to nutrient starvation, desiccation and high salt concentration) are discussed with respect to the high glycogen and trehalose contents of the cells.     

Effects of nitrogen starvation on the photosynthetic physiology of a tropical marine microalga Rhodomonas sp. (Cryptophyceae)

The effects of nitrogen starvation on biomass composition and photosynthetic function were examined in the marine cryptophyte Rhodomonas sp. Batch-cultured cells in N-sufficient medium showed a 2.5-fold increase in total carbohydrate content, and a 33% increase in cell volume when the cultures reached the stationary growth phase. These cultures also increased the ratio of phycoerythrin (PE)/hydrosoluble proteins from 6 to 22% by the 4th and 10th day of culture, respectively. In contrast, light-saturated photosynthetic activity (P_m) progressively decreased, and the value obtained at the beginning of the stationary phase was about 45% of that obtained for cells in the late exponential growth phase. Transfer to N-lacking medium caused a 3.2-fold increase in cell volume. N starvation also triggered a rapid decline in N-containing compounds such as hydrosoluble proteins and photosynthetic pigments, causing an almost complete loss of PE. The ratio of PE/hydrosoluble proteins decreased from 6 to 1% after 6 d of N deprivation. Furthermore, the PSII fluorescence capacity declined under N-starved conditions, which caused a pronounced decrease in both the P_m (circa 90%) and the apparent photosynthetic efficiency (circa 55%). Under these conditions, photosynthetically fixed carbon was used to synthesize large amounts of carbohydrates. We suggest that, in addition to the role of phycoerythrin as a light-harvesting pigment, Rhodomonas sp. responds to N-depleted conditions by mobilizing combined nitrogen from biliproteins.     

Actual and potential rates of substrate oxidation and product formation in continuous cultures ofChromatium vinosum

Kinetics of electron-donor oxidation, storage-polymer formation and growth were studied in continuous cultures ofChromatium under conditions of balanced growth as well as during transient states.Under steady-state conditions, glycogen was accumulated at all dilution rates. This observation is consistent with previously postulated ideas about an ineffective glycogen-synthesis regulation.Upon perturbing the steady states, brought about by injection of extra sulfide into steady-state cultures, the following phenomena were observed immediately, irrespective of the dilution rate: the specific rate of sulfide oxidation increased to the value found in batch cultures, the sulfur-oxidation rate was decreased, the specific glycogen-synthesis rate increased, the increment being higher the lower the dilution rate, but an increase in the specific growth rate, if any, was below the limit of detection. The inverse relationship between the specific rates of glycogen synthesis and growth after removing the substrate limitation is to be explained by a shortage of intermediates, rather than by a growth-rate dependent intrinsic glycogen-synthesis limitation, because upon complete inhibition of growth a further increase in the rate of glycogen synthesis was observed. Essayed in this way, identical glycogen-synthesis rates were found at all dilution rates.Competitive advantages of such an apparently not adapted metabolism in environments with diurnal fluctuations in substrate concentrations are discussed.Non-Standard Abbreviations N_c cell nitrogen - TS total sugar - PHB poly--hydroxybutyrate - D dilution rate - S_R reservoir concentration of the growth-limiting substrate - CAP chloramphenicol     

Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during the growth (myxamoebal) phase

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 that are grown in axenic medium containing 86mm-glucose have seven times the glycogen content of the same myxamoebae grown in the same medium but lacking added carbohydrate. 2. During the transition from the exponential to the stationary phase of growth in axenic medium containing glucose myxamoebae preferentially synthesize glycogen and can have as much as three times the glycogen content during the stationary phase as they have during the exponential phase of growth. 3. The rate of glycogen degradation by myxamoebae is, under all conditions of growth, small compared with the rate of glycogen accumulation and the changes in glycogen content thus reflect altered rates of glycogen synthesis. 4. There is no correlation between the rate of glycogen synthesis by myxamoebae and the glycogen synthetase content of the myxamoebae. 5. The activity of glycogen synthetase of D. discoideum is inhibited by a physiological concentration of ATP and this inhibition is overcome by glucose 6-phosphate. Both effects are especially marked at physiological concentrations of UDP-glucose. 6. The rate of glycogen accumulation by myxamoebae growing exponentially in axenic media can be satisfactorily accounted for in terms of the known intracellular concentrations of glucose 6-phosphate, UDP-glucose and glycogen synthetase. The rate-limiting factors controlling glycogen synthesis by the myxamoebae are apparently the substrate (UDP-glucose) and effector (glucose 6-phosphate and ATP) concentrations rather than the amount of the enzyme.     

N2-fixation in Erwinia herbicola

Four from 18 strains of Erwinia herbicola tested had nitrogenase activity and grew with N₂ as sole source of nitrogen under strict anaerobic conditions with a doubling time of 20–24 h. Nitrogenase activity started only 96–120 h after transfer to a special medium maintained under anaerobic conditions. A ten fold increase in protein per culture found after the maximum nitrogenase activity of 80–130 nmol C₂H₄. mg protein^-1·min^-1 was accompanied by a fall in pH of the medium (20 mM phosphate buffer and in 125 mM Tris-buffer) from pH 7.2 to 5.4 or less, but only to 6.8 in 100 mM phosphate buffer. In all cases we found a sharp curtailing of nitrogenase activity 48 h after the maximum. The bacteria utilized only 35–50% of the nitrogen fixed for growth. Erwinia herbicola strains differed from two strains of Enterobacter agglomerans in being unable to fix nitrogen on agar surfaces exposed to air. Specific nitrogenase activity in Erwinia herbicola is compared with data reported for other Enterobacteriaceae and is found to be higher than that reported for Klebsiella pneumoniae, Enterobacter cloacae or Citrobacter freundii.     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Cellular physiology controls photoautotrophic production of 1,2-propanediol from pools of CO2 and glycogen

Synechocystis sp. PCC 6803 PG is a cyanobacterial strain capable of synthesizing 1,2-propanediol from carbon dioxide (CO₂) via a heterologous three-step pathway and a methylglyoxal synthase (MGS) originating from Escherichia coli as an initial enzyme. The production window is restricted to the late growth and stationary phase and is apparently coupled to glycogen turnover. To understand the underlying principle of the carbon partitioning between the Calvin-Benson-Bassham (CBB) cycle and glycogen in the context of 1,2-propanediol production, experiments utilizing ¹³C labeled CO₂ have been conducted. Carbon fluxes and partitioning between biomass, storage compounds, and product have been monitored under permanent illumination as well as under dark conditions. About one-quarter of the carbon incorporated into 1,2-propanediol originated from glycogen, while the rest was derived from CO₂ fixed in the CBB cycle during product formation. Furthermore, 1,2-propanediol synthesis was depending on the availability of photosynthetic active radiation and glycogen catabolism. We postulate that the regulation of the MGS from E. coli conflicts with the heterologous reactions leading to 1,2-propanediol in Synechocystis sp. PCC 6803 PG. Additionally, homology comparison of the genomic sequence to genes encoding for the methylglyoxal bypass in E. coli suggested the existence of such a pathway also in Synechocystis sp. PCC 6803. These findings are critical for all heterologous pathways coupled to the CBB cycle intermediate dihydroxyacetone phosphate via a MGS and reveal possible engineering targets for rational strain optimization.     

Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp

The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp ^–) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp ^– cells. The agp ^– mutant had 38% less photosynthetic capacity when grown in light over 600 mol m^–2 s^–1. Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.     

Gaseous environments modify reserve carbohydrate contents and cell survival in the brewing yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The use of H₂, He and O₂ during batch fermentation of Saccharomyces cerevisiae BRAS291 increased the final intracellular glycogen contents of the cells from 2-fold to 10-fold compared with a gas-free condition, and this depended on the gas applied. Differently, the intracellular trehalose contents increased from 2-fold to 10-fold in reducing conditions compared with more oxidizing conditions. During storage at 4°C, the viability of cells cultivated with gas was twice that of cells cultivated without gas. These results could be explained by the intracellular carbohydrate contents as well as yeast ultrastructural modifications observed previously.     

Evolution of carboxylating enzymes involved in paramylon synthesis (phosphoenolpyruvate carboxylase and carboxykinase) in heterotrophically grown Euglena gracilis

Heterotrophically grown Euglena synthesize grains of paramylon, its reserve carbohydrate, in a vesicular complex of mitochondrial origin. A CO₂ fixation activity in dark grown Euglena was demonstrated in the mitochondria via paramylon. At the beginning of the exponential phase of growth, the activity of phosphoenolpyruvate carboxykinase increases before the augmentation of paramylon.At the end of the exponential phase, the activity of this enzyme decreases, and low residual levels persist in the transition and stationary phases of growth. The activity of phosphoenolpyruvate carboxylase evolves inversely during the heterotrophic growth of the algae in succinate- or a lactate-containing medium. A compartmentalized scheme of carbon metabolism in mitochondria is presented.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - PGA phosphoglyceric acid     

Pattern of inhibition of nitrate utilization by ammonium in the acidophilic thermophilic unicellular alga Cyanidium caldarium

Ammonium-induced inhibition of nitrate utilization was monitored in cell suspensions of the unicellular alga Cyanidium caldarium. It was found that the inhibition followed an exponential pattern with a t _1/2 value of about 1.5 min in cells previously grown under conditions of excess nitrate, and of about 15 min in cells grown under conditions of severe nitrate limitation. In the latter cells only, a pretreatment with cycloheximide greatly increased the t _1/2 value of inhibition. Also the resumption of nitrate utilization when ammonium was depleted followed an exponential pattern with a t _1/2 value of about 4.5 min.Our results are consistent with the hypothesis that inhibition of nitrate utilization occurs at the level of nitrate reductase activity.     

Carbohydrate formation in rewetted terrestrial cyanobacteria

Summary In the terrestrial cyanobacterium Nostoc commune Vauch. formation of carbohydrate polymers was measured upon rewetting the mats in a light-dark regime. To discriminate between carbohydrates of different physiological function, total carbohydrate was determined as anthrone-reactive material (ARM) and storage carbohydrate (glycogen) assayed by an enzymic test. In the dry thalli glycogen was found to represent less than one tenth of the ARM. After rewetting an increase of total carbohydrate was observed in illuminated samples. Only glycogen, however, showed a regular pattern of synthesis and degradation during a 12:12 h light-dark cycle. This indicates that most carbohydrates detected by anthrone belong to the metabolically inert sheath material.When illuminated colonies were kept submerged after rewetting glycogen was hydrolyzed indicative of being used in the rapid recovery of cellular functions as observed in rewetted colonies. Apparently, photosynthesis allowed for net glycogen synthesis only, provided the mats were sufficiently aerated. These findings give evidence that the (carbohydrate) sheath plays an important role in water retention in an organism bound to a terrestrial habitat.     

Glycogen in the Marine Protozoon Parauronema acutum*

SYNOPSIS. The carbohydrate which accumulates in the cytoplasm of the marine protozoon, Parauronema acutum, during normal growth was isolated, purified and characterized chemically. The highly purified material yielded only glucose residues following hydrolysis in 0.6 N HCl for 3 h at 100 C; measurement of total carbohydrate by the phenol-sulfuric acid method and by treatment with amylo-glucosidase and glucose oxidase gave similar values. Aqueous solutions of the purified material reacted with iodine to form a complex which exhibited an absorption peak at 456 nm with a shift to 484 nm in the presence of 50% saturated (NH₄)₂SO₄. Digestion with α-amylase, β-amylase, and isoamylase yielded 71%, 45% and 8.3% hydrolysis, respectively. Treatment sequentially with both isoamylase and β-amylase gave complete hydrolysis of the polymer. The average chain length (CL) determined by the isoamylase procedure was 12. These observations are consistent with the view that the carbohydrate isolated from the protozoan is a polymer consisting of α-D-glucose residues arranged in chains containing α-(1→4) linkages with branch points containing α-(1→6) linkages occurring once on the average of ～ 12 glucose residues and, as such, is indistinguishable from glycogen isolated from mammalian sources.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Evidence for two transport systems for nitrate in the acidophilic thermophilic alga Cyanidium caldarium

In the unicellular non-vacuolate red alga Cyanidium caldarium nitrate uptake occurs through two specific permease systems which, on the basis of kinetic constants can be defined as low affinity system and high affinity system. The high affinity system is saturated at very low nitrate concentrations (<1 M), whereas the low affinity system is saturated only at high nitrate concentrations (K _m=0.45±0.10 mM). The low affinity system is present in cells growing under conditions of nitrogen limitation as well as in cells growing in excess nitrate. In contrast, the high affinity system is present only in cells growing under conditions of nitrogen limitation. The high affinity system works only at acid pH and is inactive at neutral pH. The low affinity system is active both at acid and at neutral pH.     

Effects of nitrogen starvation on the photosynthetic physiology of a tropical marine microalga Rhodomonas sp. (Cryptophyceae)

The effects of nitrogen starvation on biomass composition and photosynthetic function were examined in the marine cryptophyte Rhodomonas sp. Batch-cultured cells in N-sufficient medium showed a 2.5-fold increase in total carbohydrate content, and a 33% increase in cell volume when the cultures reached the stationary growth phase. These cultures also increased the ratio of phycoerythrin (PE)/hydrosoluble proteins from 6 to 22% by the 4th and 10th day of culture, respectively. In contrast, light-saturated photosynthetic activity (P_m) progressively decreased, and the value obtained at the beginning of the stationary phase was about 45% of that obtained for cells in the late exponential growth phase. Transfer to N-lacking medium caused a 3.2-fold increase in cell volume. N starvation also triggered a rapid decline in N-containing compounds such as hydrosoluble proteins and photosynthetic pigments, causing an almost complete loss of PE. The ratio of PE/hydrosoluble proteins decreased from 6 to 1% after 6 d of N deprivation. Furthermore, the PSII fluorescence capacity declined under N-starved conditions, which caused a pronounced decrease in both the P_m (circa 90%) and the apparent photosynthetic efficiency (circa 55%). Under these conditions, photosynthetically fixed carbon was used to synthesize large amounts of carbohydrates. We suggest that, in addition to the role of phycoerythrin as a light-harvesting pigment, Rhodomonas sp. responds to N-depleted conditions by mobilizing combined nitrogen from biliproteins.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH₂PO₄), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH₂PO₄ from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO₄, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH₂PO₄, yeast autolysate (an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.