Bioenergetic characterization of transient state phosphate uptake by the cyanobacterium Anacystis nidulans

A force flow relationship based on nonequilibrium thermodynamics was derived to analyze the variable transient state phosphate uptake phenomena of cyanobacteria seen under different growth conditions and external phosphate concentrations. This relationship postulates the following basic properties of the uptake system: First, a threshold value exists, below which incorporation is energetically impossible. Second, threshold values are influenced by the activity of the phosphate uptake system, such that a decrease of the activity increases the threshold level. Third, near the thermodynamic equilibrium the uptake rate is linearly dependent on the free energy of polyphosphate formation and the pH-gradient at the thylakoid membrane. Experiments performed with Anacystis nidulans showed that phosphate uptake characteristics conformed to the properties predicted by the linear force-flow relationship. Linearity extented into regions far form thermodynamic equilibrium, e.g. to high phosphate concentrations, when algae were preconditioned to high phosphate levels. Under phosphate limited growth linearity was confined to a small concentration range, threshold values decreased below 10 nM, and the external concentration approached threshold. The data suggest that the uptake system responds to changes in the external phosphate concentration in the same way as sensory systems to input stimuli by amplifying signals and adapting to them.Abbreviations chl chlorophyll - H _e ⁺ , H _C ⁺ , H _T ⁺ protons in the external medium, the cytoplasmic and thylakoid space respectively - P_c phosphate in the cytoplasmic space - P_e phosphate in the external medium - P_n, P_n+1 polyphosphates - pH_T pH-gradient across the thylakoid membrane     

The regulation of the energy-dependent phosphate uptake by the blue-green alga Anacystis nidulans

Investigations of the energy-dependent accumulation of orthophosphate by the blue-green alga Anacystis nidulans have established: 1. The transport through the cell membrane is the rate-limiting step in the incorporation of phosphate.-2. This transport is facilitated by a carrier that can be activated by Ca²⁺ and Mg²⁺ and inhibited by EDTA.-3. The activation of the carrier in the light is associated with changes of the cytoplasmic Mg²⁺ content.-4. Intracellular phosphate is shown to be present in bound form.-5. The energy-dependent accumulation of orthophosphate within the cell depends strictly on the cytoplasmic pH and not on the energy conversion at the thylakoid membrane which is responsible for the energy supply. The cytoplasmic pH is different in the light, in the dark, and in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Orthophosphate accumulation can most readily be explained in terms of a pH dependent precipitation into a complex with bivalent cations rather than by an active transport against a concentration gradient.Abbreviation CCCP Carbonyl cyanide m-chlorophenylhydrazone     

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Summary The binding and uptake of nick-translated ³²P-labeled pBR322 by Anacystis nidulans 6301 have been characterized. Both processes were considerably enhanced in permeaplasts compared to cells. The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells. Uptake of DNA by permeaplasts was unaffected by: Mg²⁺ or Ca²⁺, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH₄Cl. ATP at 2.5–10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A. nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range. In contrast to transformation of A. nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark. The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.     

Genetic transformation of the blue-green bacterium,Anacystis nidulans

Evidence is presented which confirms the existence of genetic transformation in the blue-green bacterium, Anacystis nidulans. This process has been demonstrated for three mutations: streptomycin resistance, a phenylalanine requirement and an ornithine requirement. The optimal conditions under which transformation occurs are also investigated, and the potential of this system for genetic mapping is discussed.This work was submitted by K. G. O. in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology, Bryn Mawr College.     

Synthesis of DNA during the cell cycle of synchronous Anacystis nidulans

Cells of Anacystis nidulans strain 1402-1 incorporate [methyl-³H]thymidine or [8-³H]adenine into DNA; in synchronous cultures (21/2 h full light, 1/2 h weak light, 5 h dark), this incorporation occurs in the dark to different extents according to the labeled precursor offered or to its specific activity. The specific activity of in vivo, uniformly labeled DNA decreases to half the initial value when the cells are grown in the absence of radioactive DNA precursors during the light phase; it does not decrease during the following dark phase. If unlabeled thymidine is given during the dark phase, the specific activity of the DNA starts to decrease at the onset of the next light phase. The time course of the decrease supports the hypothesis that all cells start their DNA replication immediately after illumination and that the first cells have completed if after 1.25 h. The slowest cells then need 3.75 h for completion of DNA replication. It is discussed whether the incorporation during the dark might be due to pool size effects.     

Deposition of condensed phosphate as an effect of varying sulfur deficiency in the cyanobacterium Synechococcus sp. (Anacystis nidulans)

Uptake of orthophosphate and deposition of condensed phosphate were investigated in cells of Synechococcus sp. (Anacystis nidulans) deficient in phosphorus or sulfur. When phosphorus was restored to phosphorus-starved cells, uptake was rapid and immediate, with the greatest accumulation occurring within the first hour. Uptake was optimum in the pH 7.5–8.5 range. Long-term (6-day) studies of uptake and deposition with cells exposed to a wide range of sulfur deficiency showed that both processes were greatest when the level of exogenous sulfur was reduced to zero. The increase in cellular phosphorus as determined chemically was in agreement with the increased number and size of polyphosphate bodies at the ultrastructural level. Possible mechanisms for the control of phosphorus uptake and condensed phosphate formation by exogenous sulfur are discussed.     

The DNA,RNA and protein composition of the cyanobacterium Anacystis nidulans grown in light- and carbon dioxide-limited chemostats

The DNA, RNA and protein content of the cyanobacterium Anacystis nidulans was determined in light-limited and carbon dioxide-limited chemostat cultures over the dilution rate range, D=0.02 h^-1 to 0.19 h^-1. The macromolecular contents as a percentage of the dry weight and on a per cell basis varied significantly as a function of organism growth rate and the nature of the growth conditions. For both limitations the RNA content per cell increased [20–55 fg RNA (cell)^-1] with increasing dilution rate and also showed an increase as a percentage of the dry weight. The DNA content as a percentage of the dry weight showed a 2-fold decrease with increasing dilution rate over the range examined. On a per cell basis DNA reached a peak at D=0.1 h^-1 [4.5 fg DNA (cell)^-1] for light-limited organisms and at D=0.08 h^-1 [8.0 fg DNA (cell)^-1] for carbon dioxide-limited organisms. The q _RNA increased with increasing dilution rates over the complete growth rate range examined whilst q _DNA reached a maximum at D=0.09 to 0.10 h^-1. The protein content as a percentage of the dry weight was greater in CO₂-limited organisms than light-limited organisms but in both cultures declined as the dilution rate was increased above D=0.10 h^-1.     

Accumulation of guanosine tetraphosphate (ppGpp) under nitrogen starvation in Anacystis nidulans,a cyanobacterium

The effect of nitrate deprivation on cell growth and nucleotide level was studied in Anacystis nidulans. A 10-fold reduction in nitrate level resulted in a drastic slowdown of growth. Upon addition of nitrate to the starving cultures, after a lag period, the cells resumed growth.Nutritional shift-down induced a transitory expansion of the guanosine tetraphosphate (ppGpp) pool, preceeded by a transitory increase in GTP and ATP concentrations. After having reached peak values, the concentration of ppGpp, GTP and ATP dropped to the respective base levels. The expansion of the ppGpp pool was found to be due to an increase in ppGpp synthesis, rather than to a decrease in ppGpp breakdown. After nutritional shift-up, no decrease in the ppGpp level was found.In starving cells, a decrease in free amino acids was observed to occur concomitantly with the expansion of the ppGpp pool. The level of free amino acids started to increase simultaneously with the contraction of the ppGpp pool.     

Temperature dependent changes in absorption and fluorescence properties of the cyanobacterium Anacystis nidulans

Temperature dependent changes in absorbance and fluorescence of chlorophyll a (Chl a) were analyzed in membrane fragments and in a Chl-protein complex reconstituted with lipids isolated from the cyanobacterium Anacystis nidulans. Absorbance versus temperature curves measured at 656 nm showed an inflection point at 23–24°C and at 14–16°C in the membrane fragments prepared from A. nidulans cells, grown at 39° and 25°C, respectively. Temperature-induced absorbance changes measured at 680 and 696 nm did not show clear break points. The presence of lipids was essential in order to see a clear maximum in the fluorescence versus temperature curve of Chl a in a Chl-protein complex. It is suggested that a specific form of Chl a may be associated with lipids in the thylakoid membranes and that this form of Chl a may be responsible for temperature-induced absorbance and fluorescence yield changes in this cyanobacterium.Abbreviations Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - SDS sodium dodecyl sulphate DPB-CIW No. 802.     

Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans

The multilayered cell wall of the cyanobacterium Anacystis nidulans was studied by the freezeetching technique. A characteristic fracture face in the outer cell wall was demonstrated which is densely packed with particles of a diameter of 60–75 Å. This particle layer is comparable with layers which have been described in many cell walls of Gram-negative prokaryotes.The outer membrane of the cell wall was solubilised by extraction with phenol/water or sodium dodecyl sulfate (SDS). In the SDS-extract 31 bands were separated by polyacrylamide gel electrophoresis, among them 3–5 major proteins with molecular weights of approximately 60, 40, and 10 kdaltons, respectively. Several polypeptides of the Anacystis cell wall were comparable in their mobility with polypeptides extracted from cell walls of different Gramnegative bacteria. The analysis of the SDS-unsoluble electron dense layer (sacculi) revealed the typical components of peptidoglycan diaminopimelic acid, muramic acid, glutamic acid, glucosamine and alamine in the molar ratio of 1.0:0.9:1.1:1.5:1.9. In addition, other amino acids (molar ratio from 0.05–0.36), mannosamine (molar ratio 0.54), and lipopolysaccharide components were detected in low concentration.Abbreviations SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetate     

Mutation of the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301): Improved conditions for the isolation of auxotrophs

Previous attempts to isolate auxotrophic mutants of Anacystis nidulans produced only a limited range of phenotypes. The frequency of recovery of auxotrophic mutants has been quantified following different mutagenic and selective treatments, and their yield has been improved by using (1) a complete medium, (2) additional mutagens, (3) multiple cycles of penicillin enrichment and (4) altered pre-enrichment starvation conditions. These modified induction and selection conditions permitted the isolation of mutants defective in nitrate reductase, nitrite reductase or malate dehydrogenase, unable to reduce sulphate, or deficient in the synthesis of biotin, thiamine, paminobenzoate, serine, glutamate, adenine or uracil.     

Effect of phosphorus supply on phosphate uptake and alkaline phosphatase activity in Rhizobia

The effect of P nutrition on phosphate uptake and alkaline phosphatase activity was studied in chemostat culture for four rhizobial and three bradyrhizobial species. Phosphate-limited cells took up phosphate 10- to 180-fold faster than phosphate-rich cells. The four fast-growing rhizobial strains contained high levels of alkaline phosphatase activity under P-limited conditions compared to the repressed levels found in P-rich cells; alkaline phosphatase activity could not be detected in three slow-growing rhizobial strains, regardless of their P-status.Glycerol 1-phosphate-uptake in the cowpea Rhizobium NGR234 was derepressed over 50-fold under P-limited conditions, and appeared to be co-regulated with phosphate uptake.The phosphate-uptake system appeared similar in all strains with apparent K _m values ranging from 1.6 M to 6.0 M phosphate and maximum activities from 17.2 to 126 nmol · min^-1 · (mg dry weight of cells)^-1. Carbonyl cyanide m-chlorophenyl hydrazone strongly inhibited phosphate uptake in all strains and a number of other metabolic inhibitors also decreased phosphate uptake in the cowpea Rhizobium NGR234. The phosphate uptake system in all strains failed to catalyse exchange of ³²P label in preloaded cells or efflux of phosphate. The results suggest a single, repressible, unidirectional and energy-dependent system for the transport of phosphate into rhizobia.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulphonic acid     

Growth and photosynthetic response of the cyanobacterium Microcystis aeruginosa in relation to photoperiodicity and irradiance

Growth and photosynthetic characteristics, P _max (maximum light-saturated oxygen production rate) and (photosynthetic affinity), of Microcystis aeruginosa were studied in continuous cultures under a range of photoperiod lengths and growth irradiances. Microcystis showed a low specific maintenance rate constant and a high growth affinity for light (typical cyanobacterial features), but required a dark period to obtain maximum growth rate. P _max and per unit dry weight increased, as did pigment content, when less light became available. By regulation in and P _max (crucial in light-limiting and high-light conditions, respectively) this buoyant species can flourish in low light, but also in high-light environments which may arise when buoyancy is lost.The two different types of light conditions affected growth, and photosynthesis, in different ways. One needs thus to discriminate between photoperiod- and irradiance-limitation, which restricts the utility of simple algal growth models. It was emphasized that photosynthetic adaptation patterns of light-limited species may resemble short-term nutrient uptake kinetics of nutrient-limited organisms.With prior knowledge of the growth limitation, we were able to assess the growth rate of a natural population of Microcystis from its photosynthetic response and from data of laboratory cultures of a known physiological state.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Phytin is synthesized in the cotyledons of germinated castor-bean seeds in response to exogenously supplied phosphate

Following germination of the castor bean (Ricinus communis L.) seed, levels of phytin decline in both the endosperm and the embryo. However, as seedling growth continues, phytin increase in the latter to a level exceeding that present in the mature dry embryo, while phytin declines concomitantly in the endosperm. It is likely that phosphate mobilized from phytin in the endosperm acts as a substrate for phytin synthesis in the embryo. This is supported by the observation that isolated embryos supplied with phosphate accumulate phytin, particularly in the cotyledons. This increase is enhanced whenmyo-inositol is provided concurrently as a carbon source. Phytin synthesis in the cotyledons of the isolated embryos can occur without the attached axis. Whether initially exposed to exogenous phosphate or not, the isolated cotyledons remain competent in their ability to synthesize phytin for an extended post-germinative period, even though the major reserves are being mobilized at this time.     

Enzymes of glycogen mobilization in the photosynthetic procaryote,Anacystis nidulans

Glycogen, the principal storage compound of assimilatory products in Anacystis nidulans, is synthesized in the light and degraded in the dark. ¹⁴C-labelled glycogen and its radioactive limit dextrin obtained by phosphorylase action were used as substrates to identify enzymes involved in glycogen mobilization. A crude homogenate of cells kept in the dark contained the following enzymes: glycogen phosphorylase (EC 2.4.1.1.) that is firmly bound to glycogen, a debranching enzyme that hydrolyzes 1,6--glucosidic bonds, and an -glucosidase (EC 3.2.1.20). Other amylolytic enzymes were not detectable Using ion exchange chromatography on DEAE-cellulose, -glucosidase and the debranching enzyme could be partly separated from each other and completely from the phosphorylase-glycogen complex. On the basis of their known substrate specificities, the cooperation of these 3 enzymes is sufficient to account for the complete conversion of glycogen into glucose and glucose 1-phosphate.