Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.     

Ion Transport in Hydrodictyon africanum

The concentrations of K, Na, and Cl in the cytoplasm and vacuole, the tracer fluxes of these ions into and out of the cenocyte, and the electrical potential difference between bathing solution and vacuole and cytoplasm, have been measured in Hydrodictyon africanum. If the ions were acted on solely by passive electrochemical forces, a net efflux of K and Cl and a net influx of Na would be expected. Tracer fluxes indicate a net influx of K and Cl and efflux of Na in the light; these net fluxes are consequently active, with an obligate link to metabolism. The effects of darkness and low temperature indicate that most of the tracer K and Cl influx and Na efflux are linked to metabolism, while the corresponding tracer fluxes in the direction of the free energy gradient are not. Ouabain specifically inhibits the metabolically linked portions of tracer K influx and Na efflux. Alterations in the external K concentration have similar effects on metabolically mediated K influx and Na efflux. It would appear that K influx and Na efflux are linked, at least in the light.     

Ouabain-insensitive K influx in Hydrodictyon africanum

Summary The occurrence is reported of cells of Hydrodictyon africanum which have, contrary to previous reports by the author, a K influx which is almost insensitive to ouabain. The conditions which govern the ouabain-sensitivity of the K influx have not yet been defined. The low ouabain sensitivity of the total K influx seems to be related to a smaller than usual activity of the component of K influx which is linked to Na efflux, and also to a smaller sensitivity of this component to inhibition by ouabain. The major components of K influx in ouabain-insensitive cells correspond to those components previously described as the passive and Cl-linked components.The occurrence is also reported of an increased sensitivity of active Cl influx (and the coupled cation influxes) in the light to uncouplers when the medium is aerated or otherwise stirred.     

Energetics of Active Phosphate Influx in Hydrodictyon africanum

The energy source for active phosphate influx in Hydrodictyonafricanum has been investigated using gas mixtures with andwithout O₂ and CO₂, light of various wavelengths, and metabolicinhibitors selective for respiratory or photosynthetic electrontransport and phosphorylation. It is concluded that, as in theother green algae studied, active phosphate transport requiresATP. In the dark this is supplied by oxidative phosphorylation;in the light the influx is much less sensitive to inhibitionof oxidative phosphorylation, and photophosphorylation (includingcyclic photophosphorylation) can act as energy source. Thissituation is more like that for active K influx (coupled toactive Na efflux) than to active Cl influx in H. africanum,except that the active dark influx is relatively greater forphosphate influx. The significance of these results for themechanism of regulation of light-stimulated ATP-requiring processes,and for the role of photosynthetic and oxidative phosphorylationin the energy metabolism of green cells, is discussed.     

Uptake of Methylammonium Ions by Hydrodictyon africanum

Methylamine influx into Hydrodictyon has been measured with[^l4C]methylamine. The influx increases with rising externalpH up to about pH 8. Between pH 8 and 9 influx remains quiteconstant, with a further increase above pH 9. Influx is light-dependent,temperature-sensitive, and is decreased by . During short-term influx (less than 4 h) metabolism of methylamineappears negligible. Prolonged influx results in CH₃ accumulation, and efflux of K⁺, Na⁺, and H⁺. There is no effecton Cl^– influx. Methylamine decreases the membrane electricp.d. by 60–120 mV at external concentrations of 0?2–1?0mM. The results indicate that, below pH 9, methylamine enters thecell almost entirely as CH₃. It is suggested that a passive electrogenic uniporter is involved, and thatby analogy uniport of may also be expected in Hydrodictyon. The results are discussed in relation to theevidence for uptake of CH₃ and by other plant cells.     

Light Stimulation of Active Transport in Hydrodictyon africanum

The mechanism of light stimulation of active K and Cl influx and active Na efflux, in Hydrodictyon africanum has been investigated using different wavelengths of red light and different gas mixtures, and the inhibitors DCMU and CCCP. The active Cl influx requires photosystem 2, since its relative quantal efficiency falls with increasing wavelength of red light, and it is as sensitive to the inhibitor DCMU as is photosynthesis; it is relatively insensitive to the uncoupler CCCP. The active K influx and active Na efflux are inhibited by CCCP, but the relative quantal efficiency of these processes increases with increasing wavelength of red light, and they are relatively insensitive to DCMU. These cation fluxes can be supported by cyclic photophosphorylation, whereas Cl influx needs photosystem 2 but probably not ATP.     

Measurement of Simultaneous Oxygen Evolution and Uptake in Hydrodictyon africanum

Oxygen uptake and evolution in illuminated and darkened cellsof Hydrodictyon africanum have been measured using ¹⁸O₂ massspeetrometry. Under conditions of light and CO₂ saturation forphotosynthesis, light stimulates oxygen uptake more than two-fold.This stimulation is prevented by DCMU but is not affected bycyanide or the uncoupler CCCP. The data are consistent withthe occurrence of a pseudocyclic electron flow and photophosphorylationin vivo in H. africanum; this agrees with data on light-dependentactive phosphate influx in this alga. Part of the light-stimulatedoxygen uptake might be involved in glycolate synthesis by thepathway proposed by Coombs and Whittingham.     

The Mechanism of Photosynthetic Use of Bicarbonate by Hydrodictyon africanum

Hydrodictyon africanum can photosynthesize at high pH underconditions in which HCO₃^– rather than CO₂ is the carbonspecies entering the cell. A passive entry of HCO₃^– seemsunlikely; a metabolic HCO₃^– pump is proposed. It is possiblethat such a pump is related to a light-dependent reaction specificto the use of HCO₃^–. This reaction is dependent on photosystem2, but appears to be independent of ATP. These characteristicsare similar to those of active lightdependent Cl influx in H.africanum, and suggest a similar energy source for the two pumps.The HCO₃^– pump may be electrogenic.     

ATP Synthesis Coupled to Nitrate Photoreduction in the Alga Hydrodictyon africanum

The presence of exogenous stimulates O₂evolution in illuminated H. africanum cells at the CO₂ compensationpoint, and it is likely that reduction in the light uses reductant produced in non-cyclic electron flow.When sources of ATP other than fermentation are absent, thepresence of , light, and a functional non-cyclic electron transport pathway stimulates active, ATP-dependentH₂ influx. This is consistent with non-cyclic electron flow associated with reduction being coupled to ATP synthesis. This -dependent ATP synthesis may be quantitatively important as a source ofATP during photolithotrophic growth with as N source in H. africanum and other algal unicells.     

Measurement of Cytoplasmic pH by the DMO Technique in Hydrodictyon africanum

The 5, 5-dimethyl-[2-¹⁴C]oxazolidine-2, 4-dione (DMO) distributiontechnique for the measurement of intracellular pH has been appliedto giant cells of Hydrodictyon africanum. Significant metabolism of DMO was found in this alga; the free[DMO + DMO–] in subcellular samples is thus derived fromthe total label in cells equilibrated in [¹⁴C]DMO solutionsby measuring and subtracting the label in metabolic productsof DMO. A further problem arises from the observation that theDMO concentration in the vacuolar sap is always lower than thatpredicted by the transmembrane equilibration of undissociatedDMO from the bathing medium. This is interpreted in terms ofa finite permeability to the anion DMO–. Since the effectof P_DMO– on the DMO distribution is much smaller at thetonoplast (where the transmembrane electrical potential differenceis small) than at the plasmalemma, the values of cytoplasmicpH are computed assuming equilibration of undissociated DMOacross the tonoplast. At an external pH of 7.0 the cytoplasmic pH is about 7.4; decreaseor increase of external pH by 1 unit causes a decrease and anincrease in cytoplasmic p11 respectively of about 0.2 pH units.Determinations of _vo at pH 6, 7, and 8, together with an assumedconstant value of _cv, permit calculations of µ_H+ at theplasmalemma and tonoplast. The values are relatively independentof external pH in the range pH 6–8 at 21–25 and12–14 kJ mol^–1 respectively. The significance ofthese results for the regulation of intracellular pH, and forthe regulation and energising of the fluxes of ions, is discussed.     

Light mediated regulation of nitrate assimilation in Synechococcus leopoliensis

Synechococcus leopoliensis was cultivated in a light/dark regime of 12:12 h. After onset of the illumination (2 h), the specific activity of nitrite reductase, glutamine synthetase and isocitric dehydrogenase increased; that of glucose-6-phosphate dehydrogenase decreased and that of nitrate reductase and NAD- (NADP) glutamate dehydrogenase remained nearly unchanged.This stimulation of the enzymes in vivo was also observed in vitro. Also, when extracts from darkened cells were incubated with thioredoxin and dithioerythriol enzyme activities increased in the same amount as obtained in vivo. In addition, glucose-6-phosphate dehydrogenase and isocitric dehydrogenase were stimulated by Mn²⁺ and Mg²⁺ in the assay mixture. Glutamine synthetase activity was enhanced only by Mg²⁺ while Mn²⁺ was inhibitory.The results are discussed with respect to the regulation of nitrogen metabolism by light.Abbreviations GS glutamine synthetase - GOGAT glutamate-oxoglutarate-aminotransferase - TR thioredoxin - DTE dithioerythritol - LD change from light to dark     

Bacterial nitrate assimilation: gene distribution and regulation

In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.     

The Action of Phlorizin on Photosynthesis and Light-stimulated Ion Transport in Hydrodictyon africanum

Phlorizin at 1 mM inhibits the coupled active influx of K andefflux of Na in Hydrodictyon africanum in the light. It doesnot inhibit the coupled influx of Cl and monovalent cations,nor the passive ion fluxes. Photosynthesis can be stimulatedup to 30 per cent under light-saturated conditions. It is concludedthat the effects of phlorizin cannot be solely due to an inhibitionof the membrane ATPase, and that photophosphorylation must insome way be affected. The nature of this effect is discussedin the light of the effects of phlorizin on photophosphorylationin isolated chloroplasts.     

The Linkage of Light-stimulated Cl Influx to K and Na Influxes in Hydrodictyon africanum

There are reciprocal stimulations of Cl influx by K and Na,and of K and Na influx by Cl, in the light in Hydrodictyon africanum.The component of the K influx which stimulates, and is stimulatedby, Cl, is independent of the ouabainsensitive mechanism forK influx also found in H. africanum. The concentration dependenceof the cation effects on Cl influx and on the Cl-stimulatedportion of their own influxes are similar. The stimulation withK saturates at about 0.3 mM K; that with Na saturates at about2 mM Na. The Cl-dependent portions of the K and Na influxeshave similar responses to changes in photo-synthetic metabolism(far-red illumination, CDMU, and CCCP) as does the light-stimulatedCl influx. This suggests that Cl influx, and the Cl-stimulatedportions of K and Na influxes are both dependent on photosystem2 of photosynthesis, and are less sensitive to the uncouplerCCCP than is ¹⁴CO₂ fixation or the K-Na pump. It would thusappear that the Cl-dependent portions of the K and Na influxesin the light are linked to the cation-stimulated portion ofthe Cl influx. There is no very great change in the electricalcomponent of the inwardly directed passive driving force oncations under conditions in which Cl is being pumped comparedwith those under which it is not. It is not clear whether suchincrease in this driving force as do occur could account quantitativelyfor the increase in the cation influxes associated with Cl transport,or whether chemical coupling must be invoked. In addition tothe Cl-stimulated portions of the cation influxes, there arealso light-stimulated portions of K and Na influx which areindependent of Cl, not associated with the cation regulatingmechanism, and which seem to have a similar linkage to photosynthesisas does the Cl-K-Na pump. Since the light-stimulated portionof the K efflux appears to be similar to this portion of theK influx, these Cl-independent light-stimulated portions ofK and Na influxes are tentatively related to light-induced changesin cation permeability.     

Mutants of Azotobacter vinelandii altered in the regulation of nitrate assimilation

The two enzymes involved in the assimilatory pathway of nitrate in Azotobacter vinelandii are corregulated. Nitrate reductase and nitrite reductase are inducible by nitrate and nitrite. Ammonium represses induction by nitrate of both reductases. Repression by ammonium is higher in media containing 2-oxo-glutarate as carbon source than in media containing sucrose. Mutants in the gene ntrC lost nitrate and nitrite reductase simultaneously. Ten chlorate-resistant mutants with a new phenotype were isolated. In media without ammonium they had a normal phenotype, being sensitive to the toxic effect of chlorate. In media containing low ammonium concentrations they were resistant to chlorate. These mutants seem to be affected in the repression of nitrate and nitrite reductases by ammonium.     

Cyclic and Non-cyclic Photophosphorylation as Energy Sources for Active K Influx in Hydrodictyon africanum

Under anaerobic conditions in the light, active K influx inHydrodictyon africanum is supported by cyclic photophosphorylation.The use of selective inhibitors shows that, in the presenceof CO₂, a considerable portion of the ATP used by the K pumpis supplied by noncyclic photophosphorylation. The rest of theATP in these conditions comes from cyclic photophosphorylation.This is true under light-limiting as well as light-saturatedconditions. If non-cyclic photophosphorylation is inhibited (by removalof carbon dioxide, by the addition of cyanide which interfereswith the carboxylation reaction, or by inhibition of photosystemtwo with DCMU or supplying only far-red light), the K influxat low light intensities is stimulated, and its characteristicsbecome those of a process powered by cyclic photophosphorylationalone. These results are interpreted in terms of a competitionfor ATP between K influx and CO₂ fixation. Implicit in thisexplanation is a requirement for a switch of excitation energyabsorbed by photosystem one from cyclic photophosphorylationto non-cyclic photophosphorylation whenever conditions (presenceof CO₂and photosystem two activity) allow CO₂ fixation to occur. Further evidence for such a switch of excitation energy absorbedby photosystem one was obtained in experiments in which redand far-red light were applied separately and together. It wasfound that CO₂ fixation showed the Emerson enhancement effect,while K influx (in the presence of CO₂) shows a ‘de-enhancement’.This suggests that far-red light alone powers cyclic photophosphorylation;if red light is also present, some of the far-red quanta arediverted to non-cyclic photophosphorylation. The nature of the interaction between cyclic and non-cyclicphotophosphorylation is discussed in relation to these and otherpublished results.     

Acid-base regulation during exercise and recovery in humans.

Arterial pH, PCO2, standard bicarbonate, lactate, and ventilation were measured with a high sampling density during rest, exercise, and recovery in normal subjects performing upright cycle ergometer exercise. Three 6-min constant-work exercise tests (moderate, heavy, and very heavy) were performed by each subject. We found a small respiratory acidosis during the moderate-intensity exercise and an early respiratory acidosis followed by a metabolic acidosis for the heavy- and very-heavy-intensity exercise. During recovery, arterial pH rapidly returned to the preexercise value for the moderate-intensity work. However, arterial pH decreased further during the first 2 min of recovery for the heavy- and very-heavy-intensity work, before a slower return toward the resting values. We conclude that arterial acidosis is the consistent arterial pH reaction for moderate-, heavy-, and very-heavy-intensity cycle ergometer exercise in humans and that this acidosis is blunted but not eliminated by the ventilatory response. During recovery, the return to resting arterial pH and PCO2 and standard bicarbonate appears to be determined by the rate of lactate decline.     

Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We isolated Mu dI1734 insertion mutants of Klebsiella pneumoniae that were unable to assimilate nitrate or nitrite as the sole nitrogen source during aerobic growth (Nas- phenotype). The mutants were not altered in respiratory (anaerobic) nitrate and nitrite reduction or in general nitrogen control. The mutations were linked and thus defined a single locus (nas) containing genes required for nitrate assimilation. beta-Galactosidase synthesis in nas+/phi(nas-lacZ) merodiploid strains was induced by nitrate or nitrite and was inhibited by exogenous ammonia or by anaerobiosis. beta-Galactosidase synthesis in phi(nas-lacZ) haploid (Nas-) strains was nearly constitutive during nitrogen-limited aerobic growth and uninducible during anaerobic growth. A general nitrogen control regulatory mutation (ntrB4) allowed nitrate induction of phi(nas-lacZ) expression during anaerobic growth. This and other results suggest that the apparent anaerobic inhibition of phi(nas-lacZ) expression was due to general nitrogen control, exerted in response to ammonia generated by anaerobic (respiratory) nitrate reduction.     

Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype.