Method for determining the temporal response of microbial phosphate transport affinity.

Nutrient transport affinities of nutrient-starved microbial populations were measured as initial slopes of plots of limiting-nutrient transport rates versus extracellular limiting-nutrient concentrations. A method was devised for the determination of soluble reactive phosphate (Pi) affinity in Pi-limited continuous culture (aT), which was then used as an indicator of the effects of light/dark cycle (LD) perturbations on the temporal Pi transport abilities of three species of freshwater algae. Cell division was asynchronous for the green alga Selenastrum capricornutum grown in continuous cultures exposed to LD cycles. An apparent rhythm in aT for Pi was greatly affected by the population size parameter. Cell division was phased for the green alga Scenedesmus quadricauda grown in LD continuous culture. A rhythm in aT for Pi was not greatly affected by the biomass parameter. Cell division was also phased in LD continuous culture for the blue-green alga (cyanobacterium) Synechococcus Nägeli, but rhythms in other parameters could not be detected. Synechococcus Nägeli was an extremely efficient Pi transporter at low Pi concentrations in LD continuous culture, and so aT could not be calculated. The results demonstrate that aT is well suited to describing the temporal response of Pi transport in LD-perturbed, Pi-limited continuous culture.     

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.     

Circadian rhythms of feeding with microalgae of Ceriodaphnia quadrangula in laboratory culture

The circadian rhythm of grazing of microalgae by a laboratory culture of Ceriodaphnia quadrangula under continuous illumination was studied by continuous registration of chlorophyll fluorescence at the outlet of a flow-through cultivator. A culture of green alga Chlorella vulgaris was used as a feed. The data obtained were treated by the statistical spectral analysis. It was found that animals preliminarily grown under a 12 h light: 12 h dark regime and transferred to constant light showed two maxima in the circadian rhythm of grazing with periods of 0.7 and 1.1 h. Animals preliminarily grown under constant light showed no circadian rhythm of grazing. It was concluded that the circadian rhythm of grazing of C. quadrangula has endogenous nature and can change according to light conditions.     

Phosphorus-Limited Growth of a Green Alga and a Blue-Green Alga

The phosphorus-limited growth kinetics of the chlorophyte Scenedesmus quadricauda and the cyanophyte Synechococcus Nägeli were studied by using batch and continuous culturing techniques. The steady-state phosphate transport capability and the phosphorus storage capacity is higher in S. Nägeli than in S. quadricauda. Synechococcus Nägeli can also deplete phosphate to much lower levels than can S. quadricauda. These results, along with their morphological characteristics, were used to construct partial physiological profiles for each organism. The profiles indicate that this unicellular cyanophyte (cyanobacterium) is better suited for growth in phosphorus-limited oligotrophic niches than is this chlorophyte (green alga).     

Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum.

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available.     

Turnover of cyclic 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum. Phosphate flux in P1- and H2-limited chemostat cultures

The archaebacterium Methanobacterium thermoautotrophicum was grown at 65 degrees C in H2- and Pi-limited chemostat cultures at dilution rates corresponding to 3- and 4-h doubling times, respectively. Under these conditions the steady state concentration of cyclic 2,3-diphosphoglycerate was 44 mM in the H2-limited cells and 13 mM in the cells grown under Pi limitation. Flux of Pi into the cyclic pyrophosphate pool was estimated by two 32P-labeling procedures: approach to isotopic equilibrium and replacement of prelabeled cyclic diphosphoglycerate with unlabeled compound. The results unequivocally demonstrate turnover of the phosphoryl groups; either both phosphoryl groups of the cyclic pyrophosphate leave together or the second leaves at a faster rate. The half-life of the rate-determining step for loss of the phosphoryl groups was approximately equal to the culture doubling time. The Pi flowing into the cyclic diphosphoglycerate pool accounted for 19% of the total Pi flux into Pi-limited cells and 43% of the total for H2-limited cells. The high phosphate flux through the large cyclic diphosphoglycerate pool suggests that this molecule plays an important role in the phosphorus metabolism of this methanogen.     

Synchronous division and the pattern of diel vertical migration of Heterosigma akashiwo (Hada) Hada (Raphidophyceae) in a laboratory culture tank

Heterosigma akashiwo (Hada) Hada (Raphidophyceae) causes red tides in Osaka Bay (Japan). A clonal culture of the alga was grown in a 2 m tall culture tank on a 12: 12 LD cycle to determine patterns of vertical migration and cell division. A specific growth rate of 0.43 ln unit · day⁻¹ was obtained during complete mixing conditions. Under weakly stratified conditions (≈ΔT = 3–4°C/1.5 m), H. akashiwo in the tank grew and showed a similar pattern of vertical migration to that observed in the field for at least 6 days. Cell concentration, mean cell volume, and photosynthetic capacity, estimated by DCMU-induced fluorescence increase of H. akashiwo, were monitored in the stratified tank at 2-h intervals over 24 h at three levels in the water column. Cell ascent began shortly before the light period and vertically swimming cells were smaller in size than those sampled near the bottom of the tank. The cell division cycle and the pattern of vertical migration were phased individually by the light regime and were well synchronized with each other. This synchrony must be due to the interrelation between these two processes or the existence of a clock which controlled endogenous rhythms of both processes and was entrained by a light: dark cycle. The relative increase of fluorescence with DCMU was higher for migrating cells than for non-migrating cells.     

Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum

The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to “bypass” the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.     

The Fungicide Phosphonate Disrupts the Phosphate-Starvation Response in Brassica nigra Seedlings

The development of Brassica nigra seedlings over 20 d of growth was disrupted by the fungicide phosphonate (Phi) in a manner inversely correlated with nutritional inorganic phosphate (Pi) levels. The growth of Pi-sufficient (1.25 mM Pi) seedlings was suppressed when 10, but not 5, mM Phi was added to the nutrient medium. In contrast, the fresh weights and root:shoot ratios of Pi-limited (0.15 mM) seedlings were significantly reduced at 1.5 mM Phi, and they progressively declined to about 40% of control values as medium Phi concentration was increased to 10 mM. Intracellular Pi levels generally decreased in Phi-treated seedlings, and Phi accumulated in leaves and roots to levels up to 6- and 16-fold that of Pi in Pi-sufficient and Pi-limited plants, respectively. Extractable activities of the Pi-starvation-inducible enzymes phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase were unaltered in Pi-sufficient seedlings grown on 5 or 10 mM Phi. However, when Pi-limited seedlings were grown on 1.5 to 10 mM Phi (a) the induction of phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase activities by Pi limitation was reduced by 40 to 90%, whereas (b) soluble protein concentrations and the activities of the ATP-dependent phosphofructokinase and pyruvate kinase were unaffacted. It is concluded that Phi specifically interrupts processes involved in regulation of the Pi-starvation response in B. nigra.     

Studies on the control of cell division in a green alga,Ankistrodemus gracilids (Chlorococcales)

The proliferation patterns of the green alga,Ankistrodesmus gracilis were investigated by electron microscopy. The multichotomically dividing phase, namely the type I phase was characterized by multichotomical nuclear division, whereas the dichotomically dividing phase, i.e., the type II phase was related to dichotomical nuclear division. Type I phase was controlled by a lower cell density and a diurnal light-dark rhythm of LD 14:10. Type II phase was induced by a cell density of more than 1.0×10⁷ cells/ml. Independent of the 2 types of proliferation patterns, the diurnal light-dark rhythms related to the autospore release from a mother cell. The diurnal light-dark rhythm of LD 14∶10 induced the scattered release of autospores. On the other hand, a rhythm of LD 18∶6 induced the release of a couple of tightly adhered autospores from a mother cell which had been observed apparently as a type II cell by light microscopy.     

Effects of acute and chronic UV-B exposure on a green alga: a continuous culture study using a computer-controlled dynamic light regime

The green alga Selenastrum capricornutum was grown in a specially developed continuous culture system to study long-term effects of chronic UV-B exposure. The new system improves upon previous laboratory culture approaches. It is demonstrated that short-term experiments underestimate UV-B effects. It is also shown that photoinhibition cannot explain the effects under chronic exposure. Under both nutrient-replete and phosphorus-limiting conditions a UV-B mediated delay in cell division rate and an increase in the cellular content of proteins, carbohydrates and chlorophyll a was measured. Transition experiments showed that complete acclimatisation to UV-B took several cell cycles. DNA damage appears to be the major cause of the observed long-term UV-B effects.     

Did sulfate availability facilitate the evolutionary expansion of chlorophyll a+c phytoplankton in the oceans?

During the Mesozoic Era, dinoflagellates, coccolithophorids and diatoms became prominent primary producers in the oceans, succeeding an earlier biota in which green algae and cyanobacteria had been proportionally more abundant. This transition occurred during an interval marked by increased sulfate concentration in seawater. To test whether increasing sulfate availability facilitated the evolutionary transition in marine phytoplankton, the cyanobacterium Synechococcus sp., the green alga Tetraselmis suecica and three algae containing chlorophyll a+c (the diatom Thalassiosira weissflogii, the dinoflagellate Protoceratium reticulatum and the coccolithophorid Emiliania huxleyi) were grown in media containing 1, 5, 10, 20, or 30 mm SO(4) (2-) . The cyanobacterium and the green alga showed no growth response to varying [SO(4) (2-) ]. By contrast, the three chlorophyll a+c algae showed improved growth with higher [SO(4) (2-) ], but only up to 10 mm. The chlorophyll a+c algae, but not the green alga or cyanobacterium, also showed lower C:S with higher [SO(4) (2-) ]. When the same experiment was repeated in the presence of a ciliate predator (Euplotes sp.), T. suecica and T. weissflogii increased their specific growth rate in most treatments, whereas the growth rate of Synechococcus sp. was not affected or decreased in the presence of grazers. In a third experiment, T. suecica, T. weissflogii, P. reticulatum and Synechococcus sp. were grown in conditions approximating modern, earlier Paleozoic and Proterozoic seawater. In these treatments, sulfate availability, nitrogen source, metal availability and Pco(2) varied. Monospecific cultures exhibited their highest growth rates in the Proterozoic treatment. In mixed culture, T. weissflogii outgrew other species in modern seawater and T.suecica outgrew the others in Paleozoic water. Synechococcus sp. grew best in Proterozoic seawater, but did not outgrow eukaryotic species in any treatment. Collectively, our results suggest that secular increase in seawater [SO(4) (2-) ] may have facilitated the evolutionary expansion of chlorophyll a+c phytoplankton, but probably not to the exclusion of other biological and environmental factors.     

INTERACTIONS OF LIGHT/DARK CYCLES AND NITROGEN PULSES ON THE TIMING OF CELL DIVISION IN THE NITROGEN-LIMITED MARINE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)1

Cell division in most eukaryotic algae grown on alternating periods of light and dark (LD) is synchronized or phased so that cell division occurs only during a restricted portion of the LD cycle. However, the phase angle of the cell division gate, the time of division relative to the beginning of the light period, is known to be affected by growth conditions such as nutrient status and temperature. In this study, it is shown that the phase angle of cell division in a diatom, Cylindrotheca fusiformis Reimann and Lewin, is affected by the N-limited growth rate; cell division occurred later in the dark period (12:12 h LD cycle) when the growth rate was infradian (D = 0.42 d^?1) than when it was ultradian (D = 1.0 d^?1). Nitrogen-pulses did not affect the phase angle of the division gate, but could shift the time of peak cell division activity within the division gate. The effects, if any, of N-pulses were dependent upon the growth rate and the time of day that the pulses were administered. These responses indicate that the timing of cell division in this diatom is not determined solely by the zeitgeber from the LD cycle, but rather that a LD cycle control mechanism and a N-mediated control mechanism are both involved and are somewhat interdependent. In addition, an increase in protein was observed immediately after administering a N-pulse to C. fusiformis in the ultradian growth mode indicating that the accumulation of protein can be uncoupled from the cell division cycle.     

The S phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G₁ by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

EFFECTS OF PHOTOCYCLES AND PERIODIC AMMONIUM SUPPLY ON THREE MARINE PHYTOPLANKTON SPECIES. I. CELL DIVISION PATTERNS1

Cell division patterns in Thalassiosira weissflogii (Grun.), Hymenomonas carterae (Braarud and Fagerl), and Amphidinium carteri (Hulburl) grown in cyclostat culture were analyzed as functions of the periodic supply of light and the limiting nutrient (ammonium) and of combinations of these two factors. In all three species, division patterns were phased by light/dark cycles in N–limited as well as N–replte conditions, and also to ammonium pulses in N–limited growth in continuous light. Both the degree and timing of the cell cycle phasing varied among species. When both stimuli were present, the influence of the photocycle overrode the N–pulse stimulus in H. carterae and A. carteri. while in T. weissflogii, division was always phased by the timing of the N–pulse regardless of the phase angle between the photocycle and the pulse.     

The s phase is discrete and is controlled by the circadian clock in the marine dinoflagellate Gonyaulax polyedra

Cloned cultures of the dinoflagellate Gonyaulax polyedra grown in a 12-h light-12-h dark cycle (LD 12:12) were synchronized to the beginning of G1 by a two sequential filtration technique. After the second filtration, with the cultures growing in LD 12:12, not many cells had divided after 1 day, but approximately half underwent cell division after 2 days. Flow cytometric measurements of the cells revealed that there is one unique S phase starting about 12 h prior to cell division and lasting for less than 4 h. A majority of cells in cultures synchronized in the same way but maintained in continuous light (LL) after filtration also divided synchronously after 2 days. Just as for the cultures in LD 12:12, those in LL have a similar discrete DNA synthesis phase prior to division. It is concluded that the circadian control of cell division acts before the S phase, giving rise to a discontinuous DNA synthesis phased by the circadian clock.     

Cell Cycle-related and Endogenously Controlled Circadian Photosynthetic Rhythms in Euglena

The data presented for three strains of Euglena gracilis corroborate previous reports of a diel rhythm in photosynthetic capacity in division-synchronized cultures of this alga and extend these studies to free running, dividing and nondividing (stationary) cultures maintained in either 24-hour or 40-minute cycles of light and darkness. During synchronous growth entrained by LD: 12,12 or free running under LD: 1/3,1/3, photosynthetic CO(2) fixation was rhythmic with a period (24.0 or about 30 hours) corresponding to the period of the cell division rhythm in the population. Furthermore, the rhythm in CO(2) fixation (per cell) found in nondividing cultures maintained in LD: 12,12 persisted in LD: 1/3,1/3 for weeks with a free running, circadian period of approximately 30 hours. An endogenous, circadian rhythm in cellular chlorophyll was found to exist, independently of cell division, under both light regimens and in each individual experiment; this observation could reflect changes in the functional role of the pigment. In cultures maintained in LD: 1/3,1/3, the phase relationship between the rhythm of photosynthetic capacity and that of chlorophyll content varied, suggesting the possibility of desynchronization among circadian rhythms in a multioscillator, unicellular organism.     

NITROGEN LIMITATION EFFECTS OF DIFFERENT NITROGEN SOURCES ON NUTRITIONAL QUALITY OF TWO FRESHWATER ORGANISMS, SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE) AND SYNECHOCOCCUS SP. (CYANOPHYCEAE)

Food quality for grazers has been related to mineral (nitrogen, phosphorus) and biochemical (amino acids, fatty acids) constituents. The aim of the study was to examine the influence of different nitrogen sources on these constituents in two organisms, the green alga Scenedesmus quadricauda Turp. and the cyanobacterium Synechococcus sp., commonly used in feeding experiments. The two organisms were grown in continuous cultures at different growth rates. Nitrate or ammonium salts were used as nitrogen sources under both replete and limited conditions. Carbon content (mg·g⁻¹ dry weight) was stable in both organisms independent of nitrogen source, nitrogen limitation, and growth rate. Nitrogen content decreased with limitation and growth rate in Scenedesmus and to a lesser degree in Synechococcus , whereas changes in phosphorus content were not statistically significant. The relative proportions of amino acids (% of total amino acids) were relatively stable in both organisms, whereas the proportions of fatty acids varied with growth rate and limitation. Fatty acid content was much lower in Synechococcus than in Scenedesmus . At N limitation, polyunsaturated fatty acids (PUFAs) showed lower levels in both organisms. The change occurred in the ω3 PUFA (linolenic acid) of the green alga and in the ω6 PUFA (linoleic acid) of the cyanobacterium. The difference in the response of N limitation in the two organisms may be traced to the different composition of the chloroplast membranes (the prokaryotic way) and the microsomal membranes (the eukaryotic way) where the desaturation takes place.     

Effect of red and blue light on the timing of cyclin-dependent kinase activity and the timing of cell division in Chlamydomonas reinhardtii.

In this study, we describe the effect of red and blue light on the timing of cell division, DNA synthesis, and activity and presence of cyclin-dependent kinases (CDKs), in synchronous cultures of the unicellular green alga Chlamydomonas reinhardtii. Cell division and DNA synthesis were found to occur later in cells grown in blue or white light, than in red light. CDK-like activity, measured using a histone H1 kinase assay, correspondingly occurred later in cultures that were grown in blue light compared to cultures grown in red light. The amount of CDK-like proteins, as detected using an antibody against the PSTAIRE motif, showed a maximum during the division phase. We conclude that the mechanism that causes the delay in the timing of cell division in blue light has its action before DNA replication takes place and also precedes the increase in CDK-like activity.     

Purification of Synechococcus lividus by equilibrium centrifugation and its synchronization by differential centrifugation

A culture of the thermophilic, unicellular, blue-green alga Synechococcus lividus was freed from two thermophilic bacteria by use of equilibrium (isopycnic) centrifugation in Ficoll density gradients. After removal of the bacteria, the alga would grow only on agar plates in a high carbon dioxide atmosphere. Intermittent illumination, equilibrium centrifugation, and differential centrifugation were tested as techniques for obtaining synchronized cultures of the alga. Daughter cells selected by differential centrifugation in Ficoll density gradients yielded the best synchrony, and a cross-wall index of 85% was observed during the period of cell division.