Fine structure of developing polyphosphate bodies in a blue-green alga,Plectonema boryanum

Summary Stages in the development of polyphosphate bodies in the blue-green alga, Plectonema boryanum, grown under continuous illumination in the presence of excess phosphate, are reported. During the first stage, an electronlucent area appears near the nucleoplasm or cross walls; it gradually increases to a size approximately equal to that of the final polyphosphate body. In this area a porous structure of medium electron density develops, while simultaneously electron-dense material, interpreted as polyphosphate, is deposited in the adjacent cytoplasm. Eventually, this material seems to penetrate into the porous structure. When the polyphosphate bodies are fully formed, the surrounding cytoplasm does not contain detectable amounts of polyphosphate.The formation of polyphosphate bodies in P. boryanum is compared with that in some bacterial species.     

A study of methods for in situ X-ray energy dispersive analysis of polyphosphate bodies in Plectonema boryanum

A variety of preparative methods for in situ X-ray energy dispersive analysis were tested to determine their effects on the elemental composition of polyphosphate bodies in P. boryanum. The bodies were found to contain large amounts of P and K and small amounts of Ca and Mg. Air drying, freeze-drying and freeze-drying from a liquid nitrogen slush all gave similar results. Fixation of the cells in glutaraldehyde and/or OsO₄ resulted in loss of the K and enhancement of the Ca peak. Magnesium was lost during embedding in epoxy.     

Nitrogenase Activity and Photosynthesis in Plectonema boryanum

Nitrogen-starved Plectonema boryanum 594 cultures flushed with N(2)/CO(2) or A/CO(2) (99.7%/0.3%, vol/vol) exhibited nitrogenase activity when assayed either by acetylene reduction or hydrogen evolution. Oxygen evolution activities and phycocyanin pigments decreased sharply before and during the development of nitrogenase activity, but recovered in the N(2)/CO(2) cultures after a period of active nitrogen fixation. Under high illumination, the onset of nitrogenase activity was delayed; however, the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) eliminated this lag. Oxygen was a strong and irreversible inhibitor of nitrogenase activity at low (>0.5%) concentrations. In the dark, low oxygen tensions (0.5%) stimulated nitrogenase activity (up to 60% of that in the light), suggesting a limited but significant respiratory protection of nitrogenase at low oxygen tensions. DCMU was not a strong inhibitor of nitrogenase activity. A decrease in nitrogenase activity after a period of active nitrogen fixation was observed in the N(2)/CO(2-), but not in the A/CO(2-), flushed cultures. We suggest that this decrease in nitrogenase activity is due to exhaustion of stored substrate reserves as well as inhibition by the renewed oxygen evolution of the cultures. Repeated peaks of alternating nitrogenase activity and oxygen evolution were observed in some experiments. Our results indicate a temporal separation of these basically incompatible reactions in P. boryanum.     

Phosphate metabolism in blue-green bacteria. V. Factors affecting phosphate uptake in Plectonema boryanum.

Plectonema boryanum requires approximately 5 days of exposure to a culture medium lacking phosphorus to induce the "polyphosphate overplus" phenomenon. At pH9, phosphate uptake is greatest both from normal culture medium and from dilute salt solutions. Phosphate uptake from dilute salt solutions was greatest when Na+ or K+ are combined with Ca2+ or Ca2+ and Mg2+. Cells starved of phosphorus in the presence of a high concentration of K+ or Ca2+ in the medium, and then allowed to take up phosphorus under the same conditions, assimilate more phosphorus than with other major ions.     

Heavy metal uptake by intact cells and protoplasts of Aureobasidium pullulans

Protoplasts prepared from yeast-like cells, hyphae and chlamydospores of Aureobasidium pullulans can take up heavy metals such as Zn²⁺, Co²⁺, Cd²⁺ and Cu²⁺. In relation to intact cells, the sensitivity of protoplasts to Cu²⁺ and Cd²⁺ was increased although chlamydospore protoplasts were more tolerant than yeast-like cell protoplasts. Surface binding of metals was reduced in protoplasts as compared with intact cells and this reduction was particularly evident for chlamydospore protoplasts. At the highest concentrations used, uptake of Zn²⁺, Co²⁺ and Cd²⁺ by yeast-like cell protoplasts was greater than that observed in intact cells which may have been due to toxicity, especially for Cd²⁺, resulting in increased membrane permeability, though for Zn²⁺ and Co²⁺ some barrier effect of the cell wall could not be completely discounted. Chlamydospore protoplasts were capable of intracellular metal uptake, unlike intact chlamydospores, and for Zn²⁺, uptake appeared to be via a different system less specific than that of the other cell types. For chlamydospores, the use of protoplasts confirmed the importance of the cell wall in preventing entry of metal ions into the cell.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Ultrastructural localization of alkaline phosphatase in the blue-green bacterium Plectonema boryanum.

Histochemical techniques applied at the ultrastructural level have clearly established the periplasmic space as the site of alkaline phosphatase activity in Plectonema boryanum. Considerable enzyme activity is found after the organism is placed in a phosphate-free medium for 5 days. This activity is found only in the cellular fraction of the culture with no activity present in the culture medium. Localization of the site of enzyme activity in cells was investigated by a modification of the method of Costerton. Unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron-dense lead phosphate. The majority of activity appears associated with layer 3 (periplasmic space) of the cell wall.     

Effects of pesticides on cyanobacterium Plectonema boryanum and cyanophage LPP-1.

Cyanobacterium Plectonema boryanum IU 594 and cyanophage LPP-1 were used as indicator organisms in a bioassay of 16 pesticides. Experiments such as spot tests, disk assays, growth curves, and one-step growth experiments were used to examine the effects of pesticides on the host and virus. Also, experiments were done in which host or virus was incubated in pesticide solutions and then assayed for PFU. P. boryanum was inhibited by four herbicides: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 1,1-dimethyl-3-(alpha, alpha,alpha-trifluoro-m-tolyl)urea ( Fluometeron ), 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine), 2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine ( Ametryn ). One insecticide, 2-methyl-2-(methylthio)-propionaldehyde O-( methylcarbamoyl )oxime (Aldicarb), also inhibited the cyanobacterium. Two insecticides inactivated LPP-1, O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate (malathion) and Isotox . Isotox is a mixture of three pesticides: S-[2-( ethylsulfinyl )ethyl]O,O-dimethyl phosphorothioate ( Metasystox -R), 1-naphthyl methylcarbamate ( Sevin ) and 4,4'-dichloro-alpha- (trichloromethyl) benzhydrom ( Kelthane ). Two pesticide-resistant strains of P. boryanum were isolated against DCMU and Atrazine. These mutants showed resistance to all four herbicides, which indicates a relationship between these phototoxic chemicals. The results indicate that P. boryanum may be a useful indicator species for phototoxic agents in bioassay procedures.     

Effect of Cyanophage Infection on CO2 Photoassimilation in Plectonema boryanum

Cyanophage infection effects a rapid and complete cessation of CO₂ photoassimilation in Plectonema cells. From the amount of infected cells lysed, it was established that this phenomenon cannot be ascribed to lysis of the host cells either from within or from without. The possibility that the effect is due to nitrogen starvation, induced secondarily by cyanophage multiplication, was ruled out when it was found that nitrogen supplementation did not influence the inhibition. It is suggested that the arrest of CO₂ photoassimilation is an integral part of the cyanophage infection cycle in P. boryanum. This idea is supported by the nondependence of the cyanophage-induced inhibition on the input multiplicity, by the light requirement for the inhibition, and by the fact that infected Plectonema cells with inhibited CO₂ photo-assimilation support normal multiplication of the cyanophage. The pattern of light requirement for this viral inhibition further supports this suggestion.     

Light-induced proton translocation through thylakoid and cytoplasmic membranes of Plectonema boryanum

Light-induced fluorescence changes of 9-aminoacridine, an indicator of proton gradient in energy-transducing membranes, were studied in Plectonema boryanum and other cyanobacteria. The fluorescence changes observed in cell suspensions resulted from a superposition of fluorescence quenching and enhancement as the analysis of the kinetic data shows. Both components of the fluorescence changes are abolished by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and m-chlorocarbonylcyanide phenylhydrazone. The inhibitory effect of DCMU is removed by 2,3,5,6- or N,N,N′,N′-tetramethyl-p-phenylenediamine. The fluorescence quenching sensitive to substrates and uncouplers of the photophosphorylation is only observed in membrane vesicles obtained by osmotic shock of P. boryanum spheroplasts. Presumably, light-induced quenching of the dye fluorescence in the cells of cyanobacteria is due to the proton transport from the cytoplasm in the inner space of thylakoids while fluorescence enhancement is due to the proton efflux from the cytoplasm into the incubation medium.     

Uptake of magnesium,strontium, barium,and manganese byPlectonema boryanum (Cyanophyceae) with special reference to polyphosphate bodies

Summary The ability of a cyanobacterium to take up and concentrate metals in polyphosphate bodies was tested. It was found that Mg, Ba and Mn are taken up by 3 and 4 week old cultures ofP. boryanum and in all cases the metals were concentrated in the polyphosphate bodies. In the case of Mn it was also detected in the cytoplasm. Strontium was sometimes taken up and incorporated into dense bodies which contained Sr, high S, high K and low Ca. In a number of trials no uptake of Sr could be detected. Polyphosphate bodies, formed as a result of the overplus phenomenon did not incorporate the metal at the time they are being formed. Polyphosphate bodies from 2 and 3 week culture contain P, K, Ca and Mg while bodies from 4 week cultures also possess Zn.It is suggested that sequestering metals in polyphosphate bodies may be a significant mechanism by which heavy metals are concentrated.     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Heavy metal uptake by plankton and other seston particles

Abstract

The distribution of heavy metals Cd, Cu and Pb between the dissolved phase and the suspended matter has been studied in a stagnant fresh-water lake Zoommeer with the aim of finding a link between heavy metals and seston particles. Phytoplankton and Zooplankton were identified to species level and their density was determined. The average surface area and average volume, respectively, of each plankton species was calculated from the measured dimensions of 20–200 specimens of each species. Heavy metal concentrations in the dissolved phase and the particulate matter were determined by differential pulse anodic stripping voltammetry.

The seston particles were divided into 10 subdivisions and the total surface area and volume, respectively, of each subdivision was taken as an independent variable for the subsequent multiple regression analysis to find the possible correlations with the heavy metal concentrations. The obtained models can explain a very large part (up to 98% for Pb, 99% for Cd and 87% for Cu) of the variation in heavy metal concentration. An adsorption process appears to govern Cd and Pb uptake by Chlorophyceae and Dinophyceae. In addition, both Cd and Pb can penetrate into Chlorophyceae. In the case of Cu, a specific interaction with the Cyanophyceae has been found. In general, the uptake of heavy metals is highly specific for both the respective metal and the organism.