Heat Shock Proteins of Cyanobacterium Anacystis nidulans

Cells of Anacystis nidulans grown at 30°C were incubatedwith ¹⁴C-Chlorella protein hydrolysate at the elevated temperatures(30–55°C) and the effect of heat shock treatment onprotein synthesis was studied. Incubation temperatures higherthan 45°C caused a significant decrease in the incorporationof amino acids into proteins. Further, the heat shock treatmentinduced significant changes in the fluorographic profile ofthe newly synthesized proteins. (Received October 25, 1985; Accepted December 4, 1985)     

Glucolipid Synthesis Activities in Cytoplasmic and Thylakoid Membranes from the Cyanobacterium Anacystis nidulans

Cytoplasmic membranes (plasma membranes), thylakoid membranesand cell walls prepared from the cyanobacterium, Anacystis nidulans,were compared for UDP-glucose: l,2-diacylglycerol glucosyltransferaseactivity. When 1,2-dipalmitoylglycerol was added as a glucosylacceptor, both cytoplasmic membranes and thylakoid membranesincorporated glucose from UDP-glucose into monoglucosyl diacylglycerol,but the cell walls containing the outer membranes did not. Thecytoplasmic membranes incorporated about twice as much glucoseas the thylakoid membranes on a protein basis. These observationssuggest that in A. nidulans the UDP-glucose: 1,2-diacylglycerolglucosyltransferase participating in glucolipid biosynthesisis located in both cytoplasmic and thylakoid membranes, butnot in the outer membrane. ¹Solar Energy Research Group, The Institute of Physical andChemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan. (Received November 21, 1985; Accepted January 27, 1986)     

A Highly Active Oxygen-Evolving Photosystem II Preparation from the Cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem (PS)-II fraction has been isolated from the cyanobacterium, Anacystis nidulans R2, using an isolation buffer containing high concentrations of sucrose and salts and subsequent solubilization of the thylakoid membranes with the detergent Triton X-100. The isolated fraction had very high PSII activity (2500 micromoles O₂ per milligram chlorophyll per hour) and was largely depleted of PSI activity. Fluorescence emission spectra (77 K) and polypeptide analysis indicated that this preparation is highly enriched in PSII, but almost completely devoid of Cyt b₆-f and PSI complexes.     

Tentoxin Inhibits Both Photophosphorylation in Thylakoids and the ATPase Activity of Isolated Coupling Factor F1 from the Cyanobacterium Anacystis nidulans

Tentoxin strongly inhibited the ATPase activity of isolatedcoupling factor 1 (AF₁) from the cyanobacterium Anacystis nidulans,with 50% inhibition occurring at 0.3 µM. When thylakoidsfrom A. nidulans were preincubated with 0.3 µM tentoxinfor 30 min, photophosphorylation was inhibited by 50%. Measurementsof fluorescence from 9-aminoacridine indicated that tentoxininhibited the utilization of the proton gradient by ATP formationin thylakoids. These results indicate that tentoxin is a strongenergy-transfer inhibitor of photophosphorylation in A. nidulans.Tentoxin decreased the level of ATP in intact cells both inthe light and in darkness, its effects being much stronger inthe dark. Tentoxin at 50 µM strongly inhibited the growthof the cells. ³Present address: Corporate Research and Development Laboratory,Tonen Co. 1-3-1 Nishi-tsurugaoka, Ohi-machi, Saitama, 354 Japan ⁴Present address: Technology and Engineering Laboratories, AjinomotoCo., Inc. Suzuki-cho 1, Kawasaki, 210 Japan     

Photoinhibition and Recovery of Photosynthesis in psbA Gene-Inactivated Strains of Cyanobacterium Anacystis nidulans

The susceptibility of photosynthesis to photoinhibition and the rate of its recovery were studied in cyanobacterium Anacystis nidulans strain R2 and its two psbA gene-inactivated mutants R2S2C3 and R2K1. Changes in the fluorescence kinetics at 77K as well as the rate of O₂ evolution were measured when cells were exposed to high photosynthetic photon flux densities in the range of 0 to 2,000 micromoles per square meter per second. The R2S2C3 mutant has an active psbAI gene highly expressed under low and normal light intensities, whereas R2K1 possesses psbAII and psbAIII genes highly expressed under very high light intensities. The level of overall susceptibility of photosynthesis to photoinhibition was more pronounced in the wild type and the mutant R2S2C3 than in the mutant R2K1, especially at higher light intensities. In constrast, all three strains showed an increased but similar sensitivity to photoinhibition after addition of the translational inhibitor streptomycin; mutant R2K1 being slightly less sensitive at lower light intensities. The result is interpreted as demonstrating similar intrinsic susceptibility to photoinhibition of the two different forms of the D1 protein, form I and form II, encoded by the psbAI and psbAII/psbAIII genes, respectively. The increased resistance to photoinhibition of the R2K1 mutant was ascribed to an approximately 3 times higher rate of recovery than the wild type and the mutant R2S2C3. On the basis of our experiments we conclude that the susceptibilities to photoinhibition of the Anacystis nidulans psbA genes mutants studied are regulated mainly by modifying the rate of repair, i.e. the rate of turnover of the D1 protein.     

Membrane Development in the Cyanobacterium, Anacystis nidulans, during Recovery from Iron Starvation

Deprivation of iron from the growth medium results in physiological as well as structural changes in the unicellular cyanobacterium Anacystis nidulans R2. Important among these changes are alterations in the composition and function of the photosynthetic membranes. Room-temperature absorption spectra of iron-starved cyanobacterial cells show a chlorophyll absorption peak at 672 nanometers, 7 nanometers blue-shifted from its normal position at 679 nanometers. Iron-starved cells have decreased amounts of chlorophyll and phycobilins. Their fluorescence spectra (77K) have one prominent chlorophyll emission peak at 684 nanometers as compared to three peaks at 687, 696, and 717 nanometers from normal cells. Chlorophyll-protein analysis of iron-deprived cells indicated the absence of high molecular weight bands. Addition of iron to iron-starved cells induced a restoration process in which new components were initially synthesized and integrated into preexisting membranes; at later times, new membranes were assembled and cell division commenced. Synthesis of chlorophyll and phycocyanins started almost immediately after the addition of iron. The absorption peak slowly returned to its normal wavelength within 24 to 28 hours. The fluorescence emission spectrum at 77K changed over a period of 14 to 24 hours during which the 696- and 717-nanometer peaks grew to their normal levels, and the 684 nanometer peak moved to 687 nanometers and its relative intensity decreased to its normal level. Analysis of chlorophyll-protein complexes on polyacrylamide gels showed that high molecular weight chlorophyll-protein bands were formed during this time, and that low molecular weight bands (related to photosystem II) disappeared. The origin of the fluorescence emission at 687 and 696 nanometers is discussed in relation to the specific chlorophyll-protein complexes formed during iron reconstitution.     

蓝细菌Anacystis nidulans R-2藻胆体中43 Kd蛋白细胞定位的初步研究

高盐浓度条件下分离了蓝细菌Anacystis nidulans R-2的藻胆体,藻胆体中存在一种43kD的蛋白。Western blotting分析表明,该蛋白能与蓝细菌Fd：NADP氧还酶中FNRE占构域的抗体发生反应,解聚的藻胆体具有FNR黄递酶的活性,初步证明该43kD蛋白就是Fd：NADP氧还酶。Triton X-114分相实验表明,这种43kD的蛋白不能进入Triton X-114相。对藻胆体的部分解聚合实验表明,富含外周杆的组分中不存在43kD的蛋白。     

Nucleotide Sequence of a Putative Sensory Kinase Gene from the Cyanobacterium, Anacystis nidulans 6301

The nucleotide sequence of a 2,146 bp portion of the Anacystisnidulans (Synechococcus PCC6301) genome has been determined.This region contains an open reading frame (ORF) of 392 codons,whose predicted protein sequence shows partial homology to thoseof E. coli phoM and envZ. Hence ORF392 is suggested to be asensory kinase gene in cyanobacteria.     

Acclimation Processes in the Light-Harvesting System of the Cyanobacterium Anacystis nidulans following a Light Shift from White to Red Light

Cyanobacteria acclimate to changes in light by adjusting the amounts of different cellular compounds, for example the light-harvesting macromolecular complex. Described are the acclimatization responses in the light-harvesting system of the cyanobacterium Anacystis nidulans following a shift from high intensity, white light to low intensity, red light.

The phycocyanin and chlorophyll content and the relative amount of the two linker peptides (33 and 30 kilodaltons) in the phycobilisome were studied. Both the phycocyanin and chlorophyll content per cell increased after the shift, although the phycocyanin increased relatively more. The increase in phycocyanin was biphasic in nature, a fast initial phase and a slower second phase, while the chlorophyll increase was completed in one phase. The phycocyanin and chlorophyll responses to red light were immediate and were completed within 30 and 80 hours for chlorophyll and phycocyanin, respectively. An immediate response was also seen for the two phycobilisome linker peptides. The amount of both of them increased after the shift, although the 33 kilodalton linker peptide increased faster than the 30 kilodalton linker peptide. The increase of the content of the two linker peptides stopped when the phycocyanin increase shifted from the first to the second phase. We believe that the first phase of phycocyanin increase was due mainly to an increase in the phycobilisome size while the second phase was caused only by an increase in the amount of phycobilisomes. The termination of chlorophyll accumulation, which indicates that no further reaction center chlorophyll antennae were formed, occurred parallel to the onset of the second phase of phycocyanin accumulation.

     

Isolation and Characterization of the Cytoplasmic Membranes from the Blue-Green Alga (Cyanobacterium) Anacystis nidulans

Cells of the blue-green alga (cyanobacterium) Anacyslis nidulanswere disintegrated, and their thylakoid membranes and cytoplasmicmembranes were isolated by floatation centrifugation on a sucrosedensity gradient. Electron micrographs revealed that the cytoplasmicmembranes formed single closed vesicles having diameters of200–400 nm. These membranes contained xanthophylls asthe major constituent pigments and rß-carotene andchlorophyll a as very minor ones. The major peaks in their absorptionspectra were due to carotenoids at 435, 455 and 487 nm, witha minor one due to chlorophyll a at 673 nm. These findings areconsistent with the yellow color of the cytoplasmic membranes.The absorption spectrum of the membranes in the carotenoid regionwas markedly affected by temperature: with a decrease in temperature,the peaks at 455 and 487 nm diminished and a new peak appearedat 390 nm. (Received February 12, 1983; Accepted June 20, 1983)     

Phycobilisomes in Anacystis nidulans

Phycobilisomes were demonstrated in Anacystis nidulans by chemical and morphological studies on cells grown in red light. These cells showed a marked reduction in the chlorophyll-phycocyanin ratio owing to a decreased chlorophyll content. Granular structures of approximately 35 nm were observed throughout red light-grown cells, but were most distinct in the peripheral region. The presence of phycobilisomes in cells grown in red light as well as in cells grown in white light is supported by experiments in which glutaraldehyde was used to stabilize the attachment between the phycobiliprotein and the thylakoids, allowing the isolation of both in the same fraction by sucrose density gradient centrifugation.     

Changes in the Polypeptide Composition of the Cytoplasmic Membrane in the Cyanobacterium Anacystis nidulans during Adaptation to Low CO2 Conditions

When cells of Anacystis nidulans grown under high CO₂ conditions(3%) were transferred to low CO₂ conditions (0.05%), their abilityto transport extracellular inorganic carbon (C_i) into the cellsincreased severalfold. There was a marked increase of 42-kDapolypeptide in the cytoplasmic membranes during the adaptationto low CO₂ conditions, while no changes were observed in thepolypeptide compositions of the thylakoid membranes and cellwalls. The results suggested that the increase of the 42-kDapolypeptide during adaptation is involved in the increased abilityto transport C_i (Received January 28, 1985; Accepted May 30, 1985)     

Synchronization of Anacystis nidulans

A new technique of short alternating lightdark periods was successfully used to synchronize the blue-green alga Anacystis nidulans. Oxygen evolution during the cell cycle is characterized by a maximum in the middle of the cycle and by a minimum at the time of division, a pattern very similar to that found in synchronized green algae.     

Chloride transport in Anacystis nidulans

Summary Anacystis nidulans will take up and accumulate chloride ions. When the external concentration was 0.2 mM Cl^- the level in the cells was 2.8 mM Cl^- and under these conditions the flux across the cell surface was in the region of 10^-13equiv Cl^-·sec^-1·cm^-2. It is suggested that this Cl^- influx is active and operates against an electrochemical potential gradient estimated to be 117 mV or 2.68 kcal/mole. The uptake of ³⁶Cl was inhibited by low temperatures and there was a net loss of Cl^- from the cells with the level decreasing towards the equilibrium value as estimated from K⁺ distribution. Although the active influx of Cl^- was often stimulated by light this was not always the case. Dark storage treatment and regulation of the chlorophyll a/phycocyanin ratios as well as total pigment content of the cells did not clarify the conditions which brought about light stimulation. Moreover, the metabolic inhibitors CCCP and CMU and also the use of anaerobic conditions did not clearly indicate the relationship between the influx mechanism and light-dark metabolism and no firm conclusions could be made about the nature of the energy source. The variation in the degree of light stimulation probably reflects the fact that in this procaryotic organism the photosynthetic and respiratory units are located on the same membrane systems and are in very close proximity to the probable site of the Cl^- pump, the plasmalemma.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - CMU 3-(4-chlorophenyl)-1,1-dimethylurea     

Separation and Characterization of Thylakoid and Cell Envelope of the Blue-green Alga (Cyanobacterium) Anacystis nidulans

The thylakoid and the cell envelope of the blue-green alga Anacystisnidulans were separated by mechanical disruption of lysozyme-treatedcells followed by differential and density gradient centrifugation.The prepared envelope was composed of an outer membrane, a peptidoglycanlayer and possibly a part of the cytoplasmic membrane. The preparedthylakoid retained the size and intricate structure typicalof the thylakoid membrane of this alga. Light absorption andfluorescence spectra revealed that the envelope contained carotenoids,a pigment with an absorption maximum at 748 nm (P750), and asmall amount of pheophytin-like pigment with an absorption maximumat 673 nm. The thylakoid contained chlorophyll a and carotenoidsbut no P750. The thylakoid contained five kinds of carotenoids,the major ones being rß-carotene and zeaxanthin, whereasthe cell envelope contained two kinds of carotenoids, zeaxanthinand nostoxanthin. Four kinds of lipids, abundant in the blue-greenalgae, were present in both the thylakoid and the cell envelope.However, the content of sulfolipid was very low in the cellenvelope. The polypeptide compositions differed between thethylakoid and the cell envelope. Similarities between blue-greenalgal cells and eukaryotic chloroplasts are discussed with respectto the spectrophotometric and biochemical characteristics ofthe thylakoid and the envelope. (Received March 7, 1981; Accepted May 22, 1981)     

Cation regulation in Anacystis nidulans

Summary Anacystis nidulans accumulates K⁺ in preference to Na⁺. The majority of the internal K⁺ exchanges with ⁴²K by a first order process at rates of about 1.3 pequiv·cm^-2·sec^-1 in the light and 0.26 pequiv·cm^-2·sec^-1 in the dark. Although the K⁺/K⁺ exchange was stimulated by light and inhibited by 10^-4 M CCCP and 10^-5 M DCMU there are several indications that this cation is passively distributed in Anacystis. Inhibition of the exchange by CCCP and DCMU occurred at concentrations greater than those required to inhibit photosynthesis and the K⁺ fluxes were stimulated by low temperatures. Moreover, although valinomycin stimulated the exchange this compound did not induce a net K⁺ leak. Assuming K⁺ is passively distributed and in free solution within the cytoplasm, as indicated by osmotic studies, would imply that there is an active Na⁺ extrusion pump operating in this organism. As yet there are no firm conclusions about the nature of the energy source for this efflux pump.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea