The Intracellular pH of Clostridium paradoxum, an Anaerobic, Alkaliphilic, and Thermophilic Bacterium

When the extracellular pH was increased from 7.6 to 9.8, Clostridium paradoxum, a novel alkalithermophile, increased its pH gradient across the cell membrane ((Delta)pH, pH(infin) - pH(infout)) by as much as 1.3 U. At higher pH values (>10.0), the (Delta)pH and membrane potential ((Delta)(psi)) eventually declined, and the intracellular pH increased significantly. Growth ceased when the extracellular pH was greater than 10.2 and the intracellular pH increased to above 9.8. The membrane potential increased to 110 (plusmn) 8.6 mV at pH 9.1, but the total proton motive force ((Delta)p) declined from about 65 mV at pH 7.6 to 25 mV at pH 9.8. Between the extracellular pH of 8.0 and 10.3, the intracellular ATP concentration was around 1 mM and decreased at lower and higher pH values concomitantly with a decrease in growth rate.     

Biooxidation capacity of the extremely thermoacidophilic archaeon metallosphaera sedula under bioenergetic challenge

The biooxidation capacity of an extremely thermoacidophilic archaeon Metallosphaera sedula (DSMZ 5348) was examined under bioenergetic challenges imparted by thermal or chemical stress in regard to its potential use in microbial bioleaching processes. Within the normal growth temperature range of M. sedula (70-79 degrees C) at pH 2.0, upward temperature shifts resulted in bioleaching rates that followed an Arrhenius-like dependence. When the cells were subjected to supraoptimal temperatures through gradual thermal acclimation at 81 degrees C (Han et al., 1997), cell densities were reduced but 3 to 5 times faster specific leaching rates (Fe3+ released from iron pyrite/cell/h) could be achieved by the stressed cells compared to cells at 79 degrees C and 73 degrees C, respectively. The respiration capacity of M. sedula growing at 74 degrees C was challenged by poisoning the cells with uncouplers to generate chemical stress. When the protonophore 2,4-dinitrophenol (5-10 μM) was added to a growing culture of M. sedula on iron pyrite, there was little effect on specific leaching rates compared to a culture with no protonophore at 74 degrees C; 25 μM levels proved to be toxic to M. sedula. However, a significant stimulation in specific rate was observed when the cells were subjected to 1 μM nigericin (+135%) and 2 μM (+63%); 5 μM levels of the ionophore completely arrested cell growth. The ionophore effect was further investigated in continuous culture growing on ferrous sulfate at 74 degrees C. When 1 μM nigericin was added as a pulse to a continuous culture, a 30% increase in specific iron oxidation rate was observed for short intervals, indicating a potential positive impact on leaching when periodic chemical stress is applied. This study suggests that biooxidation rates can be increased by strategic exposure of extreme thermoacidophiles to chemical or thermal stress, and this approach should be considered for improving process performance. Copyright 1998 John Wiley & Sons, Inc.     

Acquired Thermotolerance and Stressed-Phase Growth of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula in Continuous Culture

The response of an extremely thermoacidophilic archaeon, Metallosphaera sedula (growth temperature range, 50 to 79(deg)C; optimum temperature, 74(deg)C; optimum pH, 2.0), to thermal stress was investigated by using a 10-liter continuous cultivation system. M. sedula, growing at 74(deg)C, pH 2.0, and a dilution rate of 0.04 hr(sup-1), was subjected to both abrupt and gradual temperature shifts in continuous culture to determine the responses of cell density levels and protein synthesis patterns. An abrupt temperature shift from 74 to 79(deg)C resulted in little, if any, changes in cell density and a small increase in total protein per cell. When the culture temperature was shifted further to 80.5(deg)C, cell density dropped to below 5 x 10(sup6) cells/ml from 10(sup8) cells/ml, leading to washout of the culture. Operation at this temperature and slightly higher temperatures, however, could be achieved by exposing the culture to thermal stress more gradually (0.5(deg)C increments). As a result, stable operation could be maintained at temperatures of up to 81(deg)C, and the washout temperature could be increased to 82.5(deg)C. Continuous culture operation at 81(deg)C for 100 h (stressed phase) led to an approximately sevenfold lower steady-state cell density than that observed for operation at or below 79(deg)C. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (both one and two dimensional) revealed significantly higher levels (sixfold increase) of a 66-kDa stress response protein (MseHSP60), immunologically related to Thermophilic Factor 55 from Sulfolobus shibatae (J. D. Trent, J. Osipiuk, and T. Pinkau, J. Bacteriol. 172:1478-1484, 1990). If the acclimated culture was returned to a lower temperature (i.e., 74(deg)C), the amount of MseHSP60 returned to levels observed prior to thermal acclimation. Furthermore, when the previously acclimated culture (at 81(deg)C) was shifted back from 74 to 81(deg)C, without going through gradual acclimation steps, the result was the immediate onset of washout, suggesting no residual thermotolerance. This study shows that gradual thermal acclimation of M. sedula could only extend the temperature range of stable growth for this organism by 2(deg)C above its maximal growth temperature, albeit at reduced cell densities. Also, this investigation illustrates the utility of continuous culture for characterizing heat shock response and assessing maximum growth temperatures for extremely thermophilic microorganisms.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Continuous Cultures

Rhodococcus erythropolis N1-36, a desulfurization strain, was grown in continuous culture at 10 different dilution rates with 50 (mu)M dibenzothiophene sulfone (DBTO(inf2)) as the growth-limiting nutrient. The steady-state biomass, concentrations of substrate (DBTO(inf2)) and product (monohydroxybiphenyl), saturation constant (0.39 (mu)M DBTO(inf2)), and cell yield coefficient (9 mg of biomass(middot)(mu)M(sup-1) DBTO(inf2)) were measured. Continuous cultures at five temperatures allowed calculation of activation energy (0.84 kcal(middot)mol(sup-1) [ca. 3.5 kJ(middot)mol(sup-1)]) near the optimal temperature (30(deg)C) for growth. A washout technique was used to calculate the maximum specific growth rate (0.235 h(sup-1)), a value equivalent to a minimum generation time of 2.95 h.     

Enthalpy changes in the formation of the proton electrochemical potential and its components

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.     

A thermostable glucoamylase from a thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp.: characterization and thermostability

A thermostable glucoamylase (GA) showed optimum activity at 70°C and pH 5.0. It was highly stable at pH 7.0. The half-life of the enzyme at pH 7.0 was 13, 8, and 3 h 40 min at 60, 65, and 70°C respectively. The residual activity of the enzyme sample incubated at 5 psi (110°C) for 30 min was about 32% of the control set (incubated at 4°C), while no activity was observed at 10 and 15 psi. The thermostability of the enzyme was enhanced twofold in the presence of 0.5% (w/v) starch at 5 psi. Thin-layer chromatography indicated that this enzyme is a GA.     

Efficiencies of singlet oxygen production and rate constants for oxygen quenching in the S1 state of dicyanonaphthalenes and related compounds.

The quantum yield of singlet oxygen ((1)O(2) ((1)Delta(g))) production (Phi(Delta)) in the oxygen quenching of photoexcited states for 1,2-dicyanonaphthalene (1,2-DCNN), 1,4-dicyanonaphthalene (1,4-DCNN) and 2,3-dicyanonaphthalene (2,3-DCNN) in cyclohexane, benzene, and acetonitrile was measured using a time-resolved thermal lens (TRTL) technique, in order to determine the efficiency of singlet oxygen ((1)Delta(g)) production in the first excited singlet state (S(1)), (f(Delta)(S)). The efficiencies of singlet oxygen ((1)Delta(g)) production from the lowest triplet state (T(1)), (f(Delta)(T)), were nearly unity for all DCNNs in all the solvents. The values of f(Delta)(S) were fairly large for 1,2-DCNN (0.33-0.57) and 1,4-DCNN (0.33-0.66), but were close to zero for 2,3-DCNN. Rate constants for oxygen quenching in the S(1) state (k(q)(S)) obtained for these compounds were significantly smaller than diffusion-controlled rate constants. The kinetics for processes leading to production and no production of singlet oxygen is discussed on the basis of the values of f(Delta)(S) and k(q)(S). The results obtained regarding phenanthrene (PH), 9-cyanophenanthrene (9-CNPH), pyrene (PY) and 1-cyanopyrene (1-CNPY) are also discussed.     

Convenient fluorescence-based methods to measure membrane potential and intracellular pH in the Archaeon Methanobacterium thermoautotrophicum

New and improved methods to determine the membrane potential (Delta Psi) and the Delta pH in methanogenic archaea were developed and tested in Methanobacterium thermoautotrophicum strain Delta H. The Delta pH measurements took advantage of the pH-dependent fluorescence properties of coenzyme F(420), the major intracellular electron carrier in the organism. The protonophore p-nitrophenol did not show any interference with the F(420) fluorescence spectra and was therefore suitable to equalize internal and external pH. The method developed allowed the determination of the intracellular pH with an error of less than 0.05 pH units.Membrane potentials could easily be assessed using the fluorescent probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)) with an accuracy of approximately 10 mV.Both methods were tested with cell suspensions of M. thermoautrophicum incubated at medium pH values between 5.5 and 8. It was found that Delta Psi and Delta pH values remained constant under these conditions. Membrane potentials were about -160 mV and Delta pH was kept at 0.35 pH units (inside minus outside) resulting in a total proton motive force of about -180 mV (inside negative).     

Analysis of soluble iron compounds in the bacterial oxidation of pyrite

Bacterial solubilization and oxidation of iron from pyrite were determined with the use of analytical methods which differentiated between total iron and reduced and oxidized forms of soluble iron. About 70% precipitation of Fe(III) was apparent in six-week old cultures. Yeast extract (0.2 and 2%) inhibited the bacterial oxidation of pyrite. Incomplete (∼50%) bacterial oxidation of iron dissolved from pyrite was attributed to the inhibition of the bacterial iron-oxidation at pH 1.2–1.3. Sample pretreatments with ion-exchange resins before analysis indicated that cationic iron concentration was almost identical with that of total iron in bacterial cultures; anionic forms of Fe amounted to about 7% of the total soluble iron.     

Factors that determine the plasma-membrane potential in bloodstream forms of Trypanosoma brucei.

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.     

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H(+)-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Delta(psi)) generated by the Schizosaccharomyces pombe plasma membrane H(+)-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Delta(psi) because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Delta(psi) at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H(+)-ATPase generates Delta(psi) of about 160 mV on the vesicle membrane. The generated Delta(psi) was stable at least over tens of minutes. An influence of the H(+) membrane permeability on the Delta(psi) buildup was demonstrated by manipulating the H(+) permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H(+)-ATPase-mediated Delta(psi) buildup.     

Bromate Reduction by Denitrifying Bacteria

In the presence of bromide, ozonation as applied in water treatment results in the formation of bromate, an ion with carcinogenic properties. The reduction of bromate by mixed bacterial populations as well as pure cultures was studied under laboratory conditions. Bromate was reduced to bromide by a mixed bacterial population with and without a preceding nitrate reduction step in an anaerobically incubated medium with ethanol as the energy and carbon source at 20 and 25 deg C. The predominating bacteria isolated from the batches showing bromate reduction were identified as Pseudomonas spp. Strains of Pseudomonas fluorescens reduced BrO(inf3)(sup-) to Br(sup-) but at a much lower rate than the mixed bacterial population did. Nitrate is a preferred electron acceptor for the bromate-reducing bacteria. Bromate reduction did not occur in the presence of NO(inf3)(sup-), and the rate of bromate reduction was at least 100 times lower than the rate of nitrate reduction. Bromate was completely converted to Br(sup-), indicating that intermediates, e.g., BrO(inf2)(sup-), did not accumulate during bromate reduction.     

Bacterial oxidation of ferrous iron at low temperatures

This study comprises the first report of ferrous iron oxidation by psychrotolerant, acidophilic iron-oxidizing bacteria capable of growing at 5 degrees C. Samples of mine drainage-impacted surface soils and sediments from the Norilsk mining region (Taimyr, Siberia) and Kristineberg (Skellefte district, Sweden) were inoculated into acidic ferrous sulfate media and incubated at 5 degrees C. Iron oxidation was preceded by an approximately 3-month lag period that was reduced in subsequent cultures. Three enrichment cultures were chosen for further work and one culture designated as isolate SS3 was purified by colony isolation from a Norilsk enrichment culture for determining the kinetics of iron oxidation. The 16S rRNA based phylogeny of SS3 and two other psychrotolerant cultures, SS5 from Norilsk and SK5 from Northern Sweden, was determined. Comparative analysis of amplified 16S rRNA gene sequences showed that the psychrotolerant cultures aligned within Acidithiobacillus ferrooxidans. The rate constant of iron oxidation by growing cultures of SS3 was in the range of 0.0162-0.0104 h(-1) depending on the initial pH. The oxidation kinetics followed an exponential pattern, consistent with a first order rate expression. Parallel iron oxidation by a mesophilic reference culture of Acidithiobacillus ferrooxidans was extremely slow and linear. Precipitates harvested from the 5 degrees C culture were identified by X-ray diffraction as mixtures of schwertmannite (ideal formula Fe(8)O(8)(OH)(6)SO(4)) and jarosite (KFe(3)(SO(4))(2)(OH)(6)). Jarosite was much more dominant in precipitates produced at 30 degrees C.     

Dissimilatory Nitrate Reduction in Anaerobic Sediments Leading to River Nitrite Accumulation

Recent studies on Northern Ireland rivers have shown that summer nitrite (NO(inf2)(sup-)) concentrations greatly exceed the European Union guideline of 3 (mu)g of N liter(sup-1) for rivers supporting salmonid fisheries. In fast-flowing aerobic small streams, NO(inf2)(sup-) is thought to originate from nitrification, due to the retardation of Nitrobacter strains by the presence of free ammonia. Multiple regression analyses of NO(inf2)(sup-) concentrations against water quality variables of the six major rivers of the Lough Neagh catchment in Northern Ireland, however, suggested that the high NO(inf2)(sup-) concentrations found in the summer under warm, slow-flow conditions may result from the reduction of NO(inf3)(sup-). This hypothesis was supported by field observations of weekly changes in N species. Here, reduction of NO(inf3)(sup-) was observed to occur simultaneously with elevation of NO(inf2)(sup-) levels and subsequently NH(inf4)(sup+) levels, indicating that dissimilatory NO(inf3)(sup-) reduction to NH(inf4)(sup+) (DNRA) performed by fermentative bacteria (e.g., Aeromonas and Vibrio spp.) is responsible for NO(inf2)(sup-) accumulation in these large rivers. Mechanistic studies in which (sup15)N-labelled NO(inf3)(sup-) in sediment extracts was used provided further support for this hypothesis. Maximal concentrations of NO(inf2)(sup-) accumulation (up to 1.4 mg of N liter(sup-1)) were found in sediments deeper than 6 cm associated with a high concentration of metabolizable carbon and anaerobic conditions. The (sup15)N enrichment of the NO(inf2)(sup-) was comparable to that of the NO(inf3)(sup-) pool, indicating that the NO(inf2)(sup-) was predominantly NO(inf3)(sup-) derived. There is evidence which suggests that the high NO(inf2)(sup-) concentrations observed arose from the inhibition of the DNRA NO(inf2)(sup-) reductase system by NO(inf3)(sup-).     

Production of a Novel Extracellular Polysaccharide by Lactobacillus sake 0-1 and Characterization of the Polysaccharide

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M(infm)) was determined at 6 x 10(sup6) Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s(sup-1), while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g (middot) liter(sup-1), which was obtained when L. sake 0-1 had been grown anaerobically at 20(deg)C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20(deg)C to 700 mg at 10(deg)C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g (middot) liter(sup-1) (middot) h(sup-1). EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.     

Persistent mitochondrial hyperpolarization,increased reactive oxygen intermediate production,and cytoplasmic alkalinization characterize altered IL-10 signaling in patients with systemic lupus erythematosus

Abnormal death signaling in lymphocytes of systemic lupus erythematosus (SLE) patients has been associated with elevation of the mitochondrial transmembrane potential (Delta psi(m)) and increased production of reactive oxygen intermediates (ROI). The resultant ATP depletion sensitizes T cells for necrosis that may significantly contribute to inflammation in patients with SLE. In the present study, the role of mitochondrial signal processing in T cell activation was investigated. CD3/CD28 costimulation of PBL elicited transient mitochondrial hyperpolarization and intracellular pH (pH(i)) elevation, followed by increased ROI production. Baseline Delta psi(m), ROI production, and pH(i) were elevated, while T cell activation-induced changes were blunted in 15 patients with SLE in comparison with 10 healthy donors and 10 rheumatoid arthritis patients. Similar to CD3/CD28 costimulation, treatment of control PBL with IL-3, IL-10, TGF-beta(1), and IFN-gamma led to transient Delta psi(m) elevation. IL-10 had diametrically opposing effects on mitochondrial signaling in lupus and control donors. Unlike healthy or rheumatoid arthritis PBL, cells of lupus patients were resistant to IL-10-induced mitochondrial hyperpolarization. By contrast, IL-10 enhanced ROI production and cell death in lupus PBL without affecting ROI levels and survival of control PBL. Ab-mediated IL-10 blockade or stimulation with antagonistic lymphokine IL-12 normalized baseline and CD3/CD28-induced changes in ROI production and pH(i) with no impact on Delta psi(m) of lupus PBL. The results suggest that mitochondrial hyperpolarization, increased ROI production, and cytoplasmic alkalinization play crucial roles in altered IL-10 responsiveness in SLE.     

Transmembrane electrical potential in Rickettsia prowazekii and its relationship to lysine transport.

The transmembrane electrical potential (delta psi) generated by Rickettsia prowazekii metabolizing glutamic acid or ATP was determined by flow dialysis with the lipophilic cation tetraphenylphosphonium and with lysine. At pH 7.0, the rickettsiae generated a delta psi as measured by tetraphenylphosphonium distribution of 90 mV. Under similar conditions, cells of R.prowazekii concentrated lysine to a gradient indicating a delta psi of 90 mV. Energy-starved cells of R. prowazekii were able to utilize exogenously supplied ATP as well as glutamic acid to generate a delta psi of 110 mV at pH 8.0. Lysine transport was markedly affected by environmental pH, the optimum pH ranging from 8.0 to 8.5. delta psi as measured with tetraphenyl-phosphonium was similarly affected in this system, with values ranging from 70 mV at pH 6.0 to 100 mV at pH 8.0. Respiration rates were also affected by the external pH, with a maximum rate of 28 nmol of O2 consumed per min per mg of rickettsial protein occurring at pH 8.0. The pH effects were readily reversible and with a rapid onset.     

Contribution of electric field (Delta psi) to steady-state transthylakoid proton motive force (pmf) in vitro and in vivo. control of pmf parsing into Delta psi and Delta pH by ionic strength

The observed levels of Delta G(ATP) in chloroplasts, as well as the activation behavior of the CF(1)CF(0)-ATP synthase, suggest a minimum transthylakoid proton motive force (pmf) equivalent to a Delta pH of approximately 2.5 units. If, as is commonly believed, all transthylakoid pmf is stored as Delta pH, this would indicate a lumen pH of less than approximately 5. In contrast, we have presented evidence that the pH of the thylakoid lumen does not drop below pH approximately 5.8 [Kramer, D. M., Sacksteder, C. A., and Cruz, J. A. (1999) Photosynth. Res. 60, 151-163], leading us to propose that Delta psi can contribute to steady-state pmf. In this work, it is demonstrated, through assays on isolated thylakoids and computer simulations, that thylakoids can store a substantial fraction of pmf as Delta psi, provided that the activities of ions permeable to the thylakoid membrane in the chloroplast stromal compartment are relatively low and the buffering capacity (beta) for protons of the lumen is relatively high. Measurements of the light-induced electrochromic shift (ECS) confirm the ionic strength behavior of steady-state Delta psi in isolated, partially uncoupled thylakoids. Measurements of the ECS in intact plants illuminated for 65 s were consistent with low concentrations of permeable ions and approximately 50% storage of pmf as Delta psi. We propose that the plant cell, possibly at the level of the inner chloroplast envelope, can control the parsing of pmf into Delta psi and Delta pH by regulating the ionic strength and balance of the chloroplast. In addition, this work demonstrates that, under certain conditions, the kinetics of the light-induced ECS can be used to estimate the fractions of pmf stored as Delta psi and Delta pH both in vitro and in vivo.     

Use of Radiolabelled Thymidine and Leucine To Estimate Bacterial Production in Soils from Continental Antarctica

Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41.     

Root-zone acidity and nitrogen source affects Typha latifolia L. growth and uptake kinetics of ammonium and nitrate

The NH(4)(+) and NO(3)(-) uptake kinetics by Typha latifolia L. were studied after prolonged hydroponics growth at constant pH 3.5, 5.0, 6.5 or 7.0 and with NH(4)(+) or NO(3)(-) as the sole N-source. In addition, the effects of pH and N source on H(+) extrusion and adenine nucleotide content were examined. Typha latifolia was able to grow with both N sources at near neutral pH levels, but the plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and higher affinity for uptake of inorganic nitrogen when grown on NH(4)(+). Growth almost completely stopped at pH 3.5, irrespective of N source, probably as a consequence of pH effects on plasma membrane integrity and H(+) influx into the root cells. Tissue concentrations of the major nutrients and adenine nucleotides were severely reduced at low pH, and the uptake capacity for inorganic nitrogen was low, and more so for NO(3)(-)-fed than for NH(4)(+)-fed plants. The maximum uptake rate, V(max), was highest for NH(4)(+) at pH 6.5 (30.9 micro mol h(-1) g(-1) root dry weight) and for NO(3)(-) at pH 5.0 (31.7 micro mol h(-1) g(-1) root dry weight), and less than 10% of these values at pH 3.5. The affinity for uptake as estimated by the half saturation constant, K((1/2)), was lowest at low pH for NH(4)(+) and at high pH for NO(3)(-). The changes in V(max) and K((1/2)) were thus consistent with the theory of increasing competition between cations and H(+) at low pH and between anions and OH(-) at high pH. C(min) was independent of pH, but slightly higher for NO(3)(-) than for NH(4)(+) (C(min)(NH(4)(+)) approximately 0.8 mmol m(-3); C(min)(NO(3)(-)) approximately 2.8 mmol m(-3)). The growth inhibition at low pH was probably due to a reduced nutrient uptake and a consequential limitation of growth by nutrient stress. Typha latifolia seems to be well adapted to growth in wetland soils where NH(4)(+) is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant's ability to modify pH-conditions in the rhizosphere.