High CO(2) Requiring Mutant of Anacystis nidulans R(2)

Some physiological characteristics of a mutant (E₁) of Anacystis nidulans R₂, incapable of growing at air level of CO₂, are described. E₁ is capable of accumulating inorganic carbon (C_i) internally as efficiently as the wild type (R₂). The apparent photosynthetic affinity for C_i in E₁, however, is some 1000 times lower than that of R₂. The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E₁ are similar to those observed in R₂. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis and depends on extracellular supply of Ci in the form of CO₂. The very high apparent photosynthetic K_m (CO₂) of the mutant indicate a large diffusion resistance for CO₂. Data obtained here are used to calculate the permeability coefficient for CO₂ between the bulk medium and the carboxylation site of cyanobacteria.     

Adaptation to Low CO(2) Level in a Mutant of Anacystis nidulans R(2) which Requires High CO(2) for Growth

The mutant E₁ of Anacystis nidulans R₂ requires high CO₂ concentration for growth but was able to adapt to low CO₂ concentration. This was exhibited by the increased ability to accumulate inorganic carbon within the cells and the large increase in the amount of a 42-kilodalton polypeptide located in the cytoplasmic membrane. The adaptation occurred in E₁ cells at an extracellular CO₂ concentration as high as 0.3%, which was 8 times the concentration for maximal adaptation in R₂ cells. The ability of E₁ cells to exhibit low CO₂ characteristics at a higher CO₂ concentration was attributed to lower intracellular CO₂ concentration.     

CO2 fixation and its regulation in Anacystis nidulans (Synechococcus)

Anacystis nidulans (Synechococcus) had a minimal doubling time of 5 hrs at 30 degrees C at saturating light intensity and carbon dioxide concentration. Half maximal growth rates in saturating CO2 occured at a light intensity of 0.54 mW per cm2, and there was an apparent threshold intensity of 0.13 mW per cm2 below which no growth occurred. Growth rate in saturating light was dependent on the concentration of CO2+H2CO3 in the medium, rather than on total dissolved CO2; half maximal rates were estimated at 0.1 mM CO2+H2CO3. Under saturating conditions of light and CO2, 14CO2 was fixed primarily into 3-PGA, and subsequently moved into sugar phosphates and amino acids. Incorporation into aspartate was relatively slow. CO2 fixation was strictly light-dependent. The changes in adenylate and pyridine nucleotide pools were followed in light/dark and dark/light transitions. Whereas adenylates relaxed slowly over 15-20 min to the concentrations characteristic of illuminated cells following the abrupt changes induced by darkening, the sharp drop in intracellular NADPH showed little dark recovery although rapid restoration occurred on reillumination. Other pyridine nucleotides showed no changes during these transitions. The nucleotide specificity and Km of partially purfied GAP dehydrogenase suggest a role for this enzyme in the regulation of CO2 fixation.     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

Changes in sulfate transport characteristics and protein composition of Anacystis nidulans R2 during sulfur deprivation.

Sulfur-starved cells of Anacystis nidulans have an increased capacity to take up sulfate. The apparent Vmax for sulfate uptake increased at least 10-fold after 24 h of sulfur deprivation, whereas the K1/2 remained unchanged at approximately 1.35 microM. The initial rate of sulfate uptake increased between 2 and 6 h after transfer of the cells to sulfur-free medium, in concert with elevated levels of three cytoplasmic membrane polypeptides with molecular masses of 43, 42, and 36 kilodaltons (kDa). The amounts of these polypeptides did not increase in response to nitrogen or phosphorus deprivation. A fourth cytoplasmic membrane polypeptide of 17 kDa did not appear until 24 h after transfer to sulfur-deficient medium. In the total soluble fraction, three polypeptides with masses of 36.5, 33.5, and 28.5 kDa increased dramatically in response to sulfur deprivation, but not in response to nitrogen or phosphorus deprivation. The specificity and abundance of these polypeptides indicate that they could play an important role in the response of A. nidulans to sulfur deprivation.     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.     

Photochemical Apparatus Organization in Anacystis nidulans (Cyanophyceae) : Effect of CO(2) Concentration during Cell Growth

Anacystis nidulans cells grown under high (3%) CO₂ partial pressure have greater phycocyanin to chlorophyll ratio (Phc/Chl) relative to cells grown under low (0.2%) CO₂ tension (Eley (1971) Plant Cell Physiol 12: 311-316). Absorbance difference spectrophotometry of A. nidulans thylakoid membranes in the ultraviolet (ΔA₃₂₀) and red (ΔA₇₀₀) regions of the spectrum reveal photosystem II/photosystem I (PSII/PSI) reaction center ratio (RCII/RCI) changes that parallel those of Phc/Chl. For cells growing under 3% CO₂, the Phc/Chl ratio was 0.48 and RCII/RCI = 0.40. At 0.2% CO₂, Phc/Chl = 0.38 and RCII/RCI = 0.24. Excitation of intact cells at 620 nm sensitized RCII at a rate approximately 20 times faster than that of RCI, suggesting that Phc excitation is delivered to RCII only. In the presence of DCMU, excitation at 620 nm induced single exponential RCII photoconversion kinetics, suggesting a one-to-one structural-functional correspondance between phycobilisome and PSII complex in the thylakoid membrane. Therefore, phycobilisomes may serve as microscopic markers for the presence of PSII in the photosynthetic membrane of A. nidulans. Neither the size of individual phycobilisomes nor the Chl light-harvesting antenna of PSI changed under the two different CO₂ tensions during cell growth. Our results are compatible with the hypothesis that, at low CO₂ concentrations, the greater relative amounts of PSI present may facilitate greater rates of ATP synthesis via cyclic electron flow. The additional ATP may be required for the active uptake of CO₂ under such conditions.     

Distinctive Light and CO(2)-Fixation Requirements of Nitrate and Ammonium Utilization by the Cyanobacterium Anacystis nidulans

The effect of light intensity on the rates of ammonium and nitrate uptake and of CO₂ fixation has been determined in intact Anacystis nidulans cells. Ammonium uptake became saturated at photon flux values of about 60 microeinsteins per square meter per second, whereas both nitrate uptake and CO₂ fixation reached saturation at about 250 microeinsteins per square meter per second, the rates of the two latter processes being tightly correlated at any light intensity assayed. Inhibition of ammonium assimilation resulted in the loss of correlation between CO₂ fixation and nitrate uptake, the latter process exhibiting then a reduced light requirement. The results establish a clear distinction between ammonium utilization and nitrate utilization with regard to their light requirement and to the nature of their dependence upon CO₂ fixation.     

Identification and Purification of a Derepressible Alkaline Phosphatase from Anacystis nidulans R2

We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M_r 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M_r 160,000 inhibit phosphatase activity by approximately 70%.     

Characterization of phycobilisome glycoproteins in the cyanobacterium Anacystis nidulans R2.

Concanavalin A-reactive linker and anchor subunits of phycobilisomes from Anacystis nidulans R2 (H. C. Riethman, T. P. Mawhinney, and L. A. Sherman, FEBS Lett. 215:209-214, 1987) were purified electrophoretically and analyzed for carbohydrate composition and quantity. Different quantities of glucose and N-acetylgalactosamine were found on the concanavalin A-reactive subunits analyzed. Proteolytic analysis of the purified subunits suggested that small regions of the 33- and 27-kilodalton linker polypeptides previously shown to be important for in vitro phycobilisome assembly contained the concanavalin A-reactive carbohydrates present on these subunits. The linker and anchor subunits from the morphologically different phycobilisome of Synechocystis sp. strain PCC6714 were also shown to be concanavalin A reactive. Membranes from iron-starved Anacystis nidulans, which lack assembled phycobilisomes and are associated with glycogen deposits, were shown to be depleted of linker and anchor proteins and to accumulate very large quantities of a concanavalin A-reactive, extrinsic membrane glycoprotein. We suggest that this iron stress-induced glycoprotein is associated with the glycogen deposits on the thylakoid surface and that the glycosylation of phycobilisome linker and anchor subunits is involved in the physiological regulation of phycobilisome assembly and degradation.     

Rubidium transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 is a unicellular blue-green alga. The cells will grow on Rb⁺ as a substitute for K⁺ but at a slower rate (t₂～ 15 h versus 12 h). Potassium is not, strictly speaking, an essential element for Synechococcus. Rubidium duxes (using ⁸⁶Rb⁺) are much slower than those of potassium, about 1 nmol m^?2 s^?1 in the light (0.35 mol m^?3 Rb⁺). ⁸⁶Rb⁺ fluxes in the dark are about 0.1 nmol m^?2 s^?1. These fluxes are very slow compared to those of Na⁺ and other ions. Isotopic influx of Rb⁺ can supply sufficient Rb⁺ to keep up with the demands for growth, but the net dux needed to keep up with growth in the light is a large proportion of the total observed dux. Kinetic studies of Rb⁺ uptake versus [Rb⁺] show two uptake phases consistent with a high-affinity and a low-affinity system. Both systems appear to be light-activated. Transport of Rb⁺ appears to be passive at pH_o 10 in the light and dark. There is no case for active transport of Rb⁺ at pH_o 7.5 in the light, but a marginal case for active uptake in the dark (about 3 kJ mol^?1). There is only a small effect of Na⁺ upon Rb⁺ transport. ⁸⁶Rb⁺ should not be used in place of ⁴²K⁺ in K⁺ nutrition studies as the details of Rb⁺ transport are different to those of K⁺ transport.     

Sulphate transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 (PCC 1942) actively accumulates sulphate in the light and dark. Intracellular sulphate was 1.35 ± 0.23 mol m^?3 (light) and 0.894 ± 0.152 mol m^?3 (dark) under control conditions (BG-11 media: pH_o, 7.5; [SO₄^2?]_o, 0.304 mol m^?3). The sulphate transporter is different from that found in higher plants: it appears to be an ATP-driven pump transporting one SO₄^2?/ATP [ΔμSO₄^2?_i,o=+ 27.7 ± 0.24 kJ mol^?1 (light) and + 24 ± 0.34 kj mol^?1 (dark)]. The rate of metabolism of SO₄^2?at pH_o, 7.5 was 150 ± 28 pmol m^?2 s^?1 (n = 185) in the light but only 12.8 ± 3.6 pmol m^?2 s^?1 (n = 61) in the dark. Light-driven sulphate uptake is partially inhibited by DCMU and chloramphenicol. Sulphate uptake is not linked to potassium, proton, sodium or chloride transport. The alga has a constitutive over-capacity for sulphate uptake [light (n= 105): K_m= 0.3 ± 0.1 mmol m^?3, V_max, = 1.8 ± 0.6 nmol m^?2 s^?1; dark (n= 56): K_m= 1.4 ± 0.4 mmol m^?3, V_max= 41 ± 22 pmol m^?2 s^?1]. Sulphite (SO₃^2?) was a competitive inhibitor of sulphate uptake. Selenate (SeO₄^2?) was an uncompetitive inhibitor.     

Immunological Characterization of Iron-Regulated Membrane Proteins in the Cyanobacterium Anacystis nidulans R2

Antibodies cross-reactive with specific membrane proteins were used to investigate membrane development in Anacystis nidulans R2 during recovery from iron stress. Polyclonal antibodies prepared using the iron-regulated chlorophyll (Chl)-protein CPVI-4 (HB Pakrasi, HC Riethman, LA Sherman 1985 Proc Natl Acad Sci USA 82: 6903-6907) as antigen were characterized and used to identify three iron stress-induced polypeptides of 36, 35, and 34 kilodaltons on immunoblots of polyacrylamide gels. The 34 kilodalton protein was shown to be a component of the Chlbinding CPVI-4 complex. The 36 kilodalton protein is an unrelated, intrinsic membrane protein tightly regulated by iron (designated IrpA), whereas the 35 kilodalton immunoreactive component is an extremely abundant glycoprotein (GP35). An analysis of photosystem II (PSII)-associated Chl-proteins during recovery from iron stress demonstrates that CPVI-4 is associated with most of the Chl present in iron-starved cells, whereas the PSII core polypeptides are present in very low levels; upon recovery, CPVI-4 diminishes in abundance as the relative levels of the other PSII proteins increase. The abundance of CPVI-4 in iron-stressed cells and the distribution of Chl among individual Chl-proteins during recovery suggest a possible role for CPVI-4 in the direction of membrane assembly during recovery from iron stress.     

Purification and characterization of a DNA polymerase from the cyanobacterium Anacystis nidulans R2.

A DNA polymerase has been highly purified from Anacystis nidulans R2. Electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels revealed that the final fraction contains three bands of Mr 107,000, 93,000, and 51,000, respectively. Analysis of purified DNA polymerase activity in situ indicates that of the three polypeptides the Mr 107,000 species has the catalytic activities. The native molecular weight of the enzyme was estimated by glycerol gradient sedimentation to be 100,000. The enzyme has an absolute requirement for a divalent cation. Mg2+ can be replaced with Mn2+, but the DNA polymerase is less active. Potassium chloride stimulates the enzyme, while potassium phosphate has no apparent effect. The enzyme is active over a pH range from 7.5 to 9.5 in 50mM Tris-HCl buffer. The ability of the cyanobacterial DNA polymerase to use activated DNA as a template, its associated 3'----5' and 5'----3' exonuclease activities, as well as its resistance to N-ethylmaleimide, dideoxynucleotides, arabinosyl-CTP and aphidicolin suggest a similarity between this enzyme and E. coli DNA polymerase I. This is the first characterization of a DNA polymerase from a cyanobacterium.     

Modulation of nitrate uptake in Anacystis nidulans by the balance between ammonium assimilation and CO2 fixation

Gradual inhibition of ammonium assimilation in Anacystis nidulans cells by increasing concentrations of 5-hydroxylysine resulted in a progressive enhancement of nitrate uptake. For 5-hydroxylysine-treated cells, the magnitude of the inhibition of nitrate uptake promoted by added ammonium was dependent on the ammonium assimilation capacity. In cells with a moderate ammonium assimilation activity, acceleration of CO2 fixation induced by bicarbonate addition antagonized the negative effect of ammonium, allowing full nitrate uptake activity. The results support the contention that nitrate utilization is under the feed-back control exerted by products of its own assimilation via ammonium, the inhibitory effect being potentiated by ammonium addition and alleviated by enhanced CO2 fixation. Results of amino acid analysis in cells exhibiting different capacities to utilize nitrate speak against these compounds as direct effectors of nitrate uptake.