The measurement of adenylate energy charge (AEC) in mycobacteria, including Mycobacterium leprae

Abstract The adenylate energy charge (AEC) of Mycobacterium leprae, Mycobacterium lepraemurium and the cultivable Mycobacterium smegmatis were determined following incubation in a variety of culture conditions. The AEC values for M. smegmatis were similar to those reported for other cultivable bacteria. The AEC values for M. leprae and M. lepraemurium purified from host tissue were lower than those of in vitro-grown organisms. The possible use of the AEC in in vitro studies with M. leprae is discussed.     

The trophic state of shallow lakes in The Netherlands

A study has been carried out on the development of an ecological assessment method for shallow lakes in The Netherlands. Analyses of eutrophication characteristics of 93 lakes with, in total, 127 sampling localities gave insight into some of the steering variables, such as total-P, total-N, chlorophyll-a and transparency. On the basis of phytoplankton species composition, three main groups of lakes could be distinguished. The first group, characterized by persistent filamentous Cyanobacteria, had the highest summer means of chlorophyll-a and total-P. The second group was characterized by non-persistent filamentous Cyanobacteria, occurring in low abundancies, and at lower chlorophyll-a and total-P concentrations. The third group of lakes was characterized by the absence or very low abundance of the filamentous Cyanobacteria. As a result of these analyses, criteria for the abatement of eutrophication in shallow lakes could be refined.     

The primary production of phytoplankton in Lake Vechten

The primary production of the phytoplankton of Lake Vechten (The Netherlands) (area, 4.7 ha; mean depth, 6 m), an unpolluted and stratified sandpit was investigated from 1969 to 1980 (except in 1971, 1975 and 1976) by the in situ ¹⁴C-technique. Other data collected include: solar radiation, transparency, oxygen and thermal structure. In winter and spring diatoms, Cryptophyceae and Chlorococcales were important algal groups, while in summer Dinophyceae and Chrysophyceae were important. The chlorophyll-a concentration was compared to the cellular biovolumes (= fresh weight) of the most abundant phytoplankton species. The primary production maxima occurred in winter, spring and during the summer stratification. The vertical profiles of photosynthesis exhibit light inhibition at surface to a maximum of 4 m. The maximum of zooplankton grazing in May–June caused a sharp decrease in the phytoplankton biomass and seston concentration accompanied by the highest transparency (clear water phase).The values for cellular C-fixation range from 10 to 1307 mg C · M^–2 · day^–1 (annual mean of 280 mg C · m^–2 · day^–1). High dark fixation (up to 100%) was encountered in the metalimnion and hypolimnion from July to October together with peaks of ¹⁴C-fixation due to a crowding of phytoplankton and phototrophic anoxic bacteria. Extracellular excretion by phytoplankton, investigated in 1977 to 1979, was 15% of the annual mean of the total C-fixation. The photosynthetic efficiency, turnover rates, and activity coefficients were low, particularly in the summer months when Ceratium hirundinella was predominant. The seasonal variations were controlled mainly by solar radiation and probably phosphate, the former being more important in the non-stratification period and the latter during the stratification period.     

Corn phosphoglycolate phosphatase: Modulation of activity by pyridine nucleotides and adenylate energy charge

The activity of corn phosphoglycolate phosphatase (EC 3.1.3.18), a bundle sheath chloroplastic enzyme, is modulated, in vitro, both by NADP(H) and adenylate energy charge. The Vmax of the enzyme is increased by NADP (25%) and NADPH (16%) whatever the pH used, 7.0 or 7.9 respective pH of the stroma in the dark and in the light. At both pH, the adenylate energy charge alone has a positive effect with two peaks of activation, characteristics for this enzyme, at 0.2 and a maximum at 0.8 accentuated under nonsaturating concentration of phosphoglycolate. At low energy charge, NADP(H) increased the activation with an additive effect most particularly observed at pH 7.9 under saturating phosphoglycolate concentration; at high energy charge, NADP(H) had a positive or negative effect on the activation, depending on the pH value and the concentrations of substrate and NADP(H).The ferredoxin-thioredoxin system does not regulate the activity since i) DTT addition do not have any effect, ii) the light-reconstituted system containing ferredoxin, ferredoxin-thioredoxin reductase, thioredoxins and thylakoids is not effective either. However, light-dark experiments indicate that phosphophycolate phosphatase can be subjected to a fine tuning of its activity.All these data suggest that light cannot induce a modification of the protein but could exert a tight control of its activity by the intermediate of Mg²⁺ and substrate concentrations and the levels of metabolites such as NADP(H), ATP, ADP, AMP. So, the regulation of the activity shown, in vitro, by energy charge and NADP(H) might be of physiological significance.Abbreviations AEC adenylate energy charge - DTT dithiothreitol - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FTR ferredoxin-thioredoxin reductase - NADP-MDH NADP-malate dehydrogenase - P glycolate-phosphoglycolate - P glycolate phosphatase-phosphoglycolate phosphatase - PSII photosystem II - PPDK pyruvate, Pi dikinase - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase     

Effect of temperature on the ATP pool and adenylate energy charge in Escherichia coli

The effect of cultivation temperature on the ATP pool and adenylate energy charge (EC) in Escherichia coli has been studied in both batch and continuous cultures. In batch culture, μ_max and the ATP pool increased with increasing growth temperatures between 27–42°C (from 0.26 to 0.62 h⁻¹, and from 5.1 to 8.2 nmol/mg dry wt., respectively). In continuous culture at a constant dilution rate (D = 0.2 h⁻¹), with increasing growth temperatures between 28–43°C, the ATP pool increased about 2-fold (from 4.2 to 8.1 nmol/mg dry wt) and the EC from 0.80 to 0.99.     

Changes in the adenylate energy charge and the induction of sporulation in Saccharomyces diastaticus

The induction of sporulation in yeast is generally accompanied by a sharp increase in energy metabolism which is evidenced by a rise of the adenylate energy charge by that time. The energy charge can be held at a low level by limitation of the phosphate supply in the growth medium. Ascus formation remains unaffected by this treatment. This suggests that the rise in ATP production normally encountered during early sporulation is not essential for the initiation of sporulation.     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV     

Determination of the trophic state by qualitative and quantitative phytoplankton analysis in two gravel pit lakes

The phytoplankton of two gravel pits with comparable nutrient concentrations but different chlorophyll-a concentrations and phytoplankton biovolume was investigated qualitatively and quantitatively over a period of two years. This study deals with the phytoplankton-rich ‘Rotter See’ and the phytoplankton-poor ‘Paulsmaar’, both South of Cologne. The trophic state of both lakes was determined comparatively according to Ryding &; Rast (1989) and the categories used by Brettum (1989). The results were compared with each other. The trophic lake index according to Hörnström (1981) was modified slightly and subsequently determined for both lakes. A combination of qualitative and quantitative phytoplankton analysis made a proposal of a rehabilitation for the Rotter See possible.     

A comparison of three methods of measuring phytoplankton biomass on a daily and seasonal basis

Three methods of measuring phytoplankton biomass (microscopic counting, electronic particle counting and determination of chlorophyll a concentration) were compared on both a daily (4 days) and a seasonal basis, in Charnwood Water, a small English freshwater lake. Correlations among measures were generally poor within days. However, good correlations were achieved among all methods on a seasonal basis. Seasonal correlations, in particular those between total particle numbers from particle counting and algal numbers from microscope, were affected by the degree of stability of the water column, with different relationships being found for mixed periods compared to stratified periods. These differences were related to an increased amount of particulate matter affecting the total particle numbers estimate during mixed periods. Other workers have found better relationships among these phytoplankton biomass methods within short periods, but there appears to be considerable variability among lakes. Therefore, it is recommended that the most appropriate method be evaluated on a individual lake basis, depending on the aims of the study.     

The trophic state of Lake Ladoga as indicated by late summer phytoplankton

As a part of the joint Russian-Finnish evaluation of human impact on Lake Ladoga, we studied the phytoplankton of the lake in order to find biological indicators for eutrophication. A second aim of the investigation was intercalibration of sampling and phytoplankton counting techniques between the Russian and Finnish laboratories. Phytoplankton samples were collected from 27 sampling stations in the lake and from the rivers Volkhov and Neva in 9–13 August 1993. In surface water samples the phytoplankton fresh weight biomass varied in the range 218–3575 mg m^–33. Highest biomass values were encountered in Sortavala Bay, and lowest ones in the western central part of the lake. Phytoplankton species composition varied considerably in the lake; blue-green and green algae predominated near-shore areas and Cryptophyceae in the offshore stations. Canonical correspondance analysis revealed close grouping of eutrophy indicating communities, dominated mainly by greens and blue-greens, in the most nutrient-rich parts of Lake Ladoga, the Volkhov and Svir Bays. Samples from the vicinity of the inflows of Vuoksi and Burnaya Rivers and off Pitkaranta formed a separate group, dominated by diatoms, most of which were typical to mesotrophic or eutrophy lakes. As judged by phytoplankton biomass values and chlorophyll a concentrations, Lake Ladoga may generally be classified as mesotrophic. Eutrophicated areas are found in the northern archipelago of the lake and in the areas influenced by large rivers.     

Short-term variations in the physiological state of phytoplankton [2pt] in a shallow temperate estuary

Short-term changes in the photosynthetic carbon metabolism and physiological state of phytoplankton were studied over a summer fortnight-long period in the Urdaibai estuary (Bay of Biscay) and related to observed environmental patterns. Day-to-day variability in the hydrographical and biological features of the estuary during the study period was due to changes in meteorological and tidal conditions. Phytoplankton biomass and primary production increased with the improvement of weather, i.e., light conditions, during neap tides. Thus a mixed bloom of cryptophyceans, Euglena sp., and the dinoflagellate Peridinium foliaceum developed in the middle and upper estuary. Photosynthetic responses of phytoplankton were related to the time-scale of changes in light regime. Allocation of photosynthate to major macromolecular classes (LMWM, lipid, polysaccharide, and protein), like phytoplankton biomass and primary production, showed strong spatio-temporal variability. High carbon fixation into low molecular weight metabolites was associated with growth limitation by low light. The relative incorporation of photosynthetic carbon into proteins increased at the beginning of the phytoplankton bloom but overall, it was rather constant. However, carbon allocation into storage products such us lipid or polysaccharide increased when carbon and energy produced under optimal growth conditions exceeded what could be assimilated into protein. These patterns are explained by both spatio-temporal changes in the environmental conditions and species-specific differences. In general, daily variability appeared to be more important than diurnal periodicity in the physiological responses of phytoplankton. Results from this study show that phytoplankton photosynthesis and carbon metabolism are simultaneously affected by biotic and abiotic factors, although short-term light fluctuations may have a major influence on the physiological state of phytoplankton in the Urdaibai estuary.     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

The phytoplankton of Lake Liddell,New South Wales: chlorophyll a concentrations,species seasonal succession,and covariation with nutrients

Samples of the phytoplankton in a freshwater lake, Lake Liddell, New South Wales (Lat: 32° 22 S, Long. 150° 1 E) were collected every 4 weeks between October 1987 and November 1988. Chlorophyll a concentrations ranged from 1.8 g 1^–1 to 9.1 g 1^–1 and were positively correlated with the following nutrient parameters: total and nett mass additions of nitrate/nitrite-N and total-N, total additions of Kjeldahl-N, and nett mass addition N-P ratios. There was no correlation between lake nutrient concentrations and chlorophyll a. Factors other than nutrient concentrations appeared to be effecting chlorophyll a concentrations as summer levels were low despite nutrient concentrations being at a maximum. In spring and summer the phytoplankton was dominated by chlorophytes, with dinoflagellates and diatoms most abundant in autumn. During winter cyanobacteria were the most abundant. The relative abundance of chlorophytes was positively correlated with in lake nitrate/nitrite-N concentrations whereas the relative abundance of cyanobacteria was negatively correlated with this parameter. Based on chlorophyll a concentrations and the phytoplankton flora Lake Liddell can be classified as mesotrophic.     

In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts

The role of AMP deaminase reaction in the stabilization of the adenylate energy charge was investigated using permeabilized yeast cells. The addition of P_i or Zn²⁺, which inhibits AMP deaminase, remarkably retarded the depletion of total adenylate pool and the recovery of the adenylate energy charge. Polyamine, an activator of the enzyme, decreased total adenylates, resulting in the enhanced recovery of the energy charge in situ. AMP deaminase can act as a regulatory enzyme in the system that stabilizes the adenylate energy charge in yeast cells under the conditions of severe metabolic stress.     

The adenylate energy charge in marine phytoplantkon: The effect of temperature on the physiological state of Skeletonema costatum (Grev.) Cleve

The adenylate energy charge $\text{([ATP] +}\text{1}\text{2}\text{[ADP])}\text{[0ATP+ADP+AMP]}$ was measured in axenic batch cultures of Skeletonema costatum (Grev.) Cleve at 2°, 10°, 15°, 20°, 24° and 30°C. The results suggest that this eurythermal diatom is physiologically capable of adapting to the 28 °C range of temperature with little apparent difference in the potential energy available to the cell. In N-limited continuous cultures at 15 °C, the energy charge values were lower than those observed in batch culture by 0.2, implying nutrient stress may result in decreased intracellular chemical energy. The utilization of the adenylate energy charge as an indicator of physiological state is suggested.     

The effect of nutrients on adenine nucleotide levels and the adenylate energy charge ratio in Spartina alterniflora and Spartina patens

Abstract. The response of the adenylate energy charge (AEC) ratio and the adenine nucleotide pools to nutrients was studied in two perennial marsh plant species. Adenine nucleotide levels and the AEC ratio were measured in Spartina patens (Alton) Muhl. plants which were grown in the greenhouse at various nutrient levels as well as in Spartina alterniflora Loisel. transplants removed from the field but maintained in marsh soil amended with different nutrient supplements. In addition, adenine nucleotide concentrations were measured in both species in their natural environment and compared with that of the same species grown in the greenhouse with a complement of nutrients.
The addition of nutrients stimulated an increase in the individual and total adenylate pools and the AEC ratio. Low nutrient levels resulted in extremely reduced adenylate pools. The AEC ratio was significantly affected in some instances, but did not decrease proportionately with the adenine nucleotide level and was typically maintained at values above 0.60. The adenine nucleotide concentrations measured in the leaves of both species were significantly higher in greenhouse-grown plants compared to field plants, but the AEC ratios were not significantly different.
Because the AEC ratio in plants can be significantly affected by nutrient level. AEC response in field investigations should be planned with attention to the potential effect of dissimilar nutrient levels among study sites.     

Larger adenylate energy charge and ATP/ADP ratios in aerenchymatous roots of Zea mays in anaerobic media as a consequence of improved internal oxygen transport

Internal transport of O₂ from the aerial tissues along the adventitious roots of intact maize plants was estimated by measuring the concentrations of adenine nucleotides in various zones along the root under an oxygen-free atmosphere. Young maize plants were grown in nutrient solution under conditions that either stimulated or prevented the formation of a lysigenous aerenchyma, and the roots (up to 210 mm long) were then exposed to an anaerobic (oxygen-free) nutrient solution. Aerenchymatous roots showed higher values than non-aerenchymatous ones for ATP content, adenylate energy charge and ATP/ADP ratios. We conclude that the lysigenous cortical gas spaces help maintain a high respiration rate in the tissues along the root, and in the apical zone, by improving internal transport of oxygen over distances of at least 210 mm. This contrasted sharply with the low energy status (poor O₂ transport) in non-aerenchymatous roots.Abbreviation AEC adenylate energy charge     

Mechanism of the inhibition of phloem loading by sodium sulfite: Effect of the pollutant on respiration, photosynthesis and energy charge in the leaf tissues

As in other materials, sodium sulfite (Na₂SO₃) inhibited both respiration and photosynthesis in leaf tissues of broad-bean L. cv. Aguadulce). Under our experimental conditions, photosynthesis was more sensitive (significant inhibition at 10 μM) to the pollutant than respiration (significant inhibition only for concentrations higher than 0.1 mM). Sulfite concentrations higher than 0.1 mM also caused the energy charge of leaf tissues to decline sharply. These results suggest that the long, term depolarization of the transmembrane potential difference noticed for concentrations of pollutant higher than 0.1 mM (Maurousset and Bonnemain, Physiol. Plant. 80: 233–237,1990) was mainly due to an indirect inhibition of the plasma membrane H⁺-ATPase activity following the decrease of the available level of ATP.