Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

GLUCOSE UPTAKE BY CYCLOTELLA CRYPTICA: DARK INDUCTION AND LIGHT INACTIVATION OF TRANSPORT SYSTEM1,2

Glucose transport capacity of C. cryptica increases in an exponential manner over 24 hr after transfer of the cells from light to complete darkness with little simultaneous increase in cell number. The transport system is rapidly inactivated when cells are transferred back to continuous light. Most of the inactivation takes place while there is still little changes in cell number. When grown on a continuous light regime, the capacity for glucose transport per cell depends on the light intensity. At intensities sufficient to saturate photosynthesis the glucose transport system is only about 5% that of dark-grown cells, while cells grown at intensities close to the light compensation point have about 30% of the capacity of dark-grown cells. The action spectrum for inactivation of glucose transport is identical to that for photosynthesis. Cells, whether grown under continuous light, in the dark in the presence of glucose, or kept in the dark without glucose, contain high levels of glucokinase and phosphofructokinase. The glucose transport system is highly specific for glucose; only galactose inhibits the uptake of glucose by about 50% when present at 10 times the concentration of glucose. The glucokinase is even more specific for glucose and is not inhibited by galactose. The phosphofructokinase is inhibited by high concentrations of ATP in cells grown under all conditions. cycloheximide inhibits the induction of glucose transport in the dark, but not the inactivation of the system in the light.     

KINETICS OF GLUCOSE TRANSPORT AND GROWTH OF CYCLOTELLA CRYPTICA REIMANN,LEWIN AND GUILLARD1,2

Growth rate as well as rate of glucose uptake of C. cryptica depends on glucose concentration in the medium according to saturation kinetics. The K _g for growth is 1.9 × 10^?5 M, and the K _t, for glucose transport is 5.8 × 10^?5 M. The maximum growth rate in the dark on glucose is considerably slower than the light-saturated growth rate at the same temperature, and does not appear to be determined by the capacity of the cell for glucose uptake. The glucose transport process is highly specific, and depends on energy metabolism. The Q ¹⁰ for the process is 2.2 (15–2.5 C). Glucose taken up by the cells is almost, quantitatively phosphorylated within 10 min, either through the transport process itself or by a high affinity kinase system in the cells.     

Evidence for Photorespiration in Tropical Grasses

Sugarcane leaves respired in full light and the CO₂ evolved could be detected in sorghum or miaze photosynthesizing in the same closed system. A combination of radiometric and infra-red gas analysis techniques allowed the estimation of photorespiration (total CO₂ evolution in light) and photosynthesis at increasing light intensities and of dark respiration. Rates of CO₂ evolution approaching those of temperate zone plants occurred at lower light intensities but rapidly decreased with higher light. Smaller but significant quantities of ¹⁴CO₂ were released even at intensities approximating full sunlight in leaves of maize, sorghum and sugarcane. Highly efficient CO₂ capture may explain the low rates of photorespiration at high light intensities.     

THE UPTAKE OF GLUCOSE BY CHLAMYDOMONAS SP.12

The glucose uptake of a species of Chlamydomonas was studied at various concentrations of d -glucose plus glucose-1-¹⁴C (0.003–10.0 mg/liter) and at various light levels (0–220 ft-c). The alga grows at 4 C either in the light or in the dark with added glucose, cellobiose, maltose, or fructose. Uptake of glucose could be described by the Michaelis-Menten equation, and both the maximum velocity of uptake and the half-saturation constant increased when the cells were exposed to glucose in the dark. However, the high value of the half-saturation constant (5 mg glucose/liter) compared with the low levels of glucose in nature (5–10 μg/liter) makes it unlikely that a transport system is effective under natural conditions. Even if a total of 10.0 mg/liter of glucose plus other organic compounds were available as substrate, the rate of photosynthesis would still be more than 10 times higher (at 220 ft-c) than the rate of organic substrate uptake. Light had no effect on the total uptake of glucose but did reduce the percentage of ¹⁴CO₂ evolved from 61% of the total ¹⁴CO taken up in the dark to 0% at 220 ft-c. This decrease could be due to either preferential use of the ¹⁴CO₂ in photosynthesis or of the photosynthate in respiration.     

Transport kinetics of maltotriose in strains ofSaccharomyces

Summary Maltotriose transport was studied in two brewer's yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized¹⁴C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasK _m values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withK _i values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.     

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO₃⁻. The V_max for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks.Additionally, we studied the regulation of root NO₃⁻ uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO₃⁻ and down-regulated by the N status and by NO₃⁻ itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status.The use of various metabolic uncouplers or inhibitors indicated that NO₃⁻ net uptake mediated by iHATS and LATS was an active transport system in both rootstocks.Key Words: Citrus, inducible high affinity transport system (iHATS), constitutive high affinity transport system (cHATS), nitrate uptake, regulation     

Photosynthesis and respiration of Griffithsia monilis (Rhodophyceae): Effect of light,salinity, and oxygen

The giant-celled alga Griffithsia monilis has a low light compensation point and saturates photosynthesis at 60–90 mol photons m^-2s^-1 (oxygen evolution and CO₂ fixation). Under dark and low light intensities ¹⁴C is preferentially incorporated into amino acids (mainly aspartate and alanine). With increasing light a gradual change was observed and, under light saturation, compounds of the anionic fraction (digeneaside and hexosephosphates) were the most strongly labeled compounds, together with the amino acids glycine and serine. To a large extent (30–40% of the total) ¹⁴C was fixed into EtOH-insoluble products, the hydrolysates of which consisted mainly of glucose and mannose. In the steady state the rates of photosynthesis and respiration decreased with increasing salinity. Changes in the rates after hyperosmotic shocks were less severe in cells adapted to high salinities. Photorespiration exists in Griffithsia: Glycine and serine are the major labeled compounds in O₂-saturated media.     

HCO3− and CO2 leakage from Synechococcus UTEX 625

The leakage of various inorganic carbon species from air-grown cells of Synechococcus UTEX 625 was investigated after a light to dark transition or during a light period using a mass spectrometer under a wide variety of experimental conditions. Total inorganic carbon efflux and CO₂ efflux during the initial period of darkness were measured with or without carbonic anhydrase in the reaction medium respectively. The HCO₃^? efflux after a light to dark transition was estimated by difference. Carbon dioxide efflux in the light was measured by inhibiting CO₂ transport with either Na₂S or COS₃ or quenching the ¹³C inorganic carbon transport by the addition of ¹²C inorganic carbon in excess. In cells in which CO₂ fixation was inhibited, when only the HCO₃^? transport system was fully operative, CO₂ effluxed continuously during the light period at a rate equal to about 25% of that in darkness. When only the CO₂ transport system was operative, HCO₃^? effluxed during the light period. The difference between the light and dark efflux rates was consistent with a 0.6 unit decrease in the intracellular pH upon darkening the cells. The permeabilities of the cell for CO₂ (2.94 ± 0.14 ± 10^?8ms^?1; mean ± SE, n=137) and HCO₃^? (1.4–1.7 ± 10^?9 ms^?1) were calculated.     

Use of glucose biosensors to measure extracellular glucose exudation by intertidal microphytobenthos in southern Tasmania

Micro glucose biosensors were used to measure net extracellular glucose produced by natural microphytobenthos and three diatom cultures (Amphora coffeaeformis, Navicula menisculus, Nitzschia longissima) from southern Tasmania, Australia. They were exposed to a light gradient in either nutrient‐replete or nutrient‐limiting conditions. Glucose exudation in the natural communities increased with increased light but the response in the cultures was variable. Similarly, nutrient‐replete conditions elicited lower rates of glucose exudation in the natural communities but produced variable species‐specific responses in the cultures. Increased glucose exudation mostly correlated with a reduction in maximum quantum yield (F_v/F_m). The same trend was observed in the natural communities for relative maximum electron transfer rates (rETR_max) but responses in the cultures were again variable and species‐specific. Responses of the three species to increased light and nutrient deficiency were variable, although glucose exudation, F_v/F_m and rETR_max was mostly lower in the nutrient‐limited media. In a second set of experiments species/communities were treated with/without antibiotics. In the dark, glucose concentrations in treatments with antibiotics remained unchanged, while in those with bacteria, it fell rapidly. In the sediment communities, glucose consumption in the dark was ~25% the rate of exudation at the highest light level. In culture, exudation rates were up to 100% greater than those with active bacteria. Rates of glucose consumption in the dark in the antibiotic–treated samples were negligible and up to 10⁴ times lower than those with active bacteria. These results demonstrate the important role extracellular glucose exudation has on maintaining an active microbial loop.     

Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 mol O₂ g^-1 fresh weight (FW) h^-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 mol O₂ g^-1 FW h^-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O₂ uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 mol glucose g^-1 FW h^-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.Abbreviation FW fresh weight - LD long day - SD short day     

H2-Photoproduction by green algae: The significance of anaerobic pre-incubation periods and of high light intensities for H2-photoproductivity ofChlorella fusca

Using sodium-dithionite as an oxygen scavenger, the influences of different light intensities and periods of anaerobic pre-incubation in the dark on H₂-photoproductivity were studied with the green algaChlorella fusca. By measuring hydrogen production in the light using manometric and gas chromatographic methods the effectiveness of sodium dithionite in stabilizing photoproduction was established. For high rates of H₂-photoproduction high light intensities up to 30,000 lux (580 W m^-2) were necessary; these are comparable to those required for light saturation of oxygen photoproduction by this alga. AlthoughChlorella fusca produces H₂ immediately after transition to anaerobic conditions, the optimum rate of H₂ production was reached after a 5 h dark adaptation period only. The results obtained are discussed with respect to characteristics of H₂-photoproduction by green algae: the initial burst kinetics, the light saturation, and the obligate period of anaerobic adaptation. It is concluded that H₂-photoproduction byChlorella is an anaerobic photosynthetic process which occurs in the absence of CO₂ and can be experimentally stabilized by exogenous oxygen scavengers.Abbreviations DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) - HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)     

High-affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes

2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na⁺-independent glucose transport system with a K_m, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with K_i's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity K_m, of 75 mM, where the K_l, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.     

The cupric ion as an inhibitor of photosynthetic electron transport in isolated chloroplasts

Strong inhibition of uncoupled photosynthetic electron transport by Cu²⁺ in isolated spinach chloroplasts was observed by measuring changes in O₂ concentration in the reaction medium. Inhibition was dependent not only on the concentration of the inhibitor, but also on the ratio of chlorophyll to inhibitor. Binding of Cu²⁺ to the chloroplast membranes resulted in removal of Cu²⁺ from solution. When chloroplasts were exposed to preincubation in light, there was increased inhibition as a result of Cu²⁺ binding to inhibitory sites. Preincubation in the dark resulted in Cu²⁺ binding to noninhibitory sites and decreased inhibition. The degree of inhibition was lower at low light intensities than at high light intensities.     

Ocean acidification effects on calcification and dissolution in tropical reef macroalgae

Net calcification rates for coral reef and other calcifiers have been shown to decline as ocean acidification (OA) occurs. However, the role of calcium carbonate dissolution in lowering net calcification rates is unclear. The objective of this study was to distinguish OA effects on calcification and dissolution rates in dominant calcifying macroalgae of the Florida Reef Tract, including two rhodophytes (Neogoniolithon strictum, Jania adhaerens) and two chlorophytes (Halimeda scabra, Udotea luna). Two experiments were conducted: (1) to assess the difference in gross (⁴⁵Ca uptake) versus net (total alkalinity anomaly) calcification rates in the light/dark and (2) to determine dark dissolution (⁴⁵CaCO₃), using pH levels predicted for the year 2100 and ambient pH. At low pH in the light, all species maintained gross calcification rates and most sustained net calcification rates relative to controls. Net calcification rates in the dark were ~84% lower than in the light. In contrast to the light, all species had lower net calcification rates in the dark at low pH with chlorophytes exhibiting net dissolution. These data are supported by the relationship (R² = 0.82) between increasing total alkalinity and loss of ⁴⁵Ca from pre-labelled ⁴⁵CaCO₃ thalli at low pH in the dark. Dark dissolution of ⁴⁵CaCO₃-labelled thalli was ~18% higher in chlorophytes than rhodophytes at ambient pH, and ~ twofold higher at low pH. Only Udotea, which exhibited dissolution in the light, also had lower daily calcification rates integrated over 24 h. Thus, if tropical macroalgae can maintain high calcification rates in the light, lower net calcification rates in the dark from dissolution may not compromise daily calcification rates. However, if organismal dissolution in the dark is additive to sedimentary carbonate losses, reef dissolution may be amplified under OA and contribute to erosion of the Florida Reef Tract and other reefs that exhibit net dissolution.

     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Sulphate uptake in the unicellular marine red algaRhodella maculata

Sulphate uptake by the unicellular marine red algaRhodella maculata conforms to Michaelis-Menten kinetics. Two uptake systems have been found: a low affinity system with an apparentK _m of 22 mM, and a high affinity system with an apparentK _m of 63.4 M. Transition from the low to the high affinity system can occur within 2.5 min, in response to a decrease in the ambient sulphate concentration to below 10 mM. Assimilation rates in the dark are about 20% those in the light, although enhancement by light is independent of the quanlity of light supplied above 27 mol m^-2 s^-1. Use of metabolic inhibitors indicates that photophosphorylation provides the main source of energy for sulphate assimilation, through both cyclic and non-cyclic electron flow.Abbreviations used APS-kinase ATP:adenylyl-sulphate 3-phosphotransferase (E.C. 2.7.1.25) - ATP-sulphurylase ATP:sulphate adenylyltransferase (E.C.2.7.74) - DCMU [3-(3,4-dichlorophenyl)]-1,1 dimethylurea - 2,4 DNP 2,4-dinitrophenol - DBMIB Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone)     

COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

Growth characteristics of flagellated cells of Phaeocystis pouchetii revealed by diel changes in cellular DNA content

The present study reports on effects of different light:dark periods, light intensities, N:P ratios and temperature on the specific growth rate of flagellated cells of Phaeocystis pouchetii in culture. The specific growth rate was estimated by diel changes in cellular DNA content. The cellular DNA content and cell cycle of flagellated cells of P. pouchetii are shown, and the importance of light:dark period in cell division is demonstrated. Diel patterns of the cellular DNA content showed that cell division was confined to the dark period. The cells dealt with more than one division per day by rapid divisions shortly after each other.The specific growth rates (μ_DNA) based on the DNA cell cycle model were in close agreement with specific growth rates (μ_Cell) determined from cell counts. The temperature affected the specific growth rates (multiple regression, p < 0.01) and were higher at 5 °C (μ ≤ 2.2 d⁻¹) than at 10 °C (μ ≤1.6 d⁻¹). Increasing the light:dark period from 12:12 h to 20:4 h affected the specific growth rate of P. pouchetii at the lower temperature tested (5 °C) (multiple regression, p < 0.01), resulting in higher specific growth rates than at 10 °C. At 10 °C, the effect of light:dark period was severely reduced. Neither light nor nutrients could compensate the reduction in specific growth rates caused by elevated temperature. The specific growth rates was not affected by the N:P ratios tested (multiple regression, p = 0.21). The experiments strongly suggest that the flagellated cells have a great growth potential and could play a dominating role in northern areas at increased day length.     

Characteristics of mixotrophic growth of Synechocystis sp. in an enclosed photobioreactor

Synechocystis sp. PCC 6803 was grown in a 2.5 l enclosed photobioreactor on medium with or without glucose. The incident light intensities ranged from 1.5 klux to 7 klux. The highest average specific growth rates of mixotrophic culture and photoautotrophic culture were, respectively, 1.3 h^–1 at a light intensity of 7 klux on 3.2 g l^–1 glucose and 0.3 h^–1 at both light intensities of 5 klux and 7 klux. The highest cell density 2.5 g l ^–1 was obtained at both of light intensities 5 klux and 7 klux on 3.2 g glucose l^–1. Glucose consumption decreased with decreasing light intensity. The energy yields of mixotrophic cultures were 4 to 6 times higher than that of photoautotrophic cultures. Light favored mixotrophic growth of Synechocystis sp. PCC 6803, especially at higher light intensities (5–7 klux).