María Isabel Orús

Biogenesis and Ultrastructure of Carboxysomes from Wild Type and Mutants of Synechococcus sp. Strain PCC 7942

Immature inclusions representing three progressive steps of carboxysome biogenesis have been identified in Synechococcus during the period of adaptation to low-CO2 conditions: (a) ring-shaped structures, (b) electron-translucent inclusions with the shape of a carboxysome and the internal orderly arrangement of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) molecules, and (c) carboxysomes with an internal electron-translucent area, which seem to be the penultimate stage of carboxysome maturation. The ability to build up normal carboxysomes is impaired in three (M3, EK6, and D4) of four high-carbon-requiring mutants studied in this work. M3 and EK6 exhibit abundant immature electron-translucent carboxysomes but no mature ones. This finding supports the contention that an open reading frame located 7.5 kb upstream of the gene encoding the large subunit of Rubisco (altered in M3) is involved in the carboxysome composition and confirms the structural role of the small subunit of Rubisco (slightly modified in EK6) in the assembly of these structures. D4 shows few typical carboxysomes and frequent immature types, its genetic lesion affecting the apparently unrelated gene encoding a subunit of phosphoribosyl aminoamidazole carboxylase of the purine biosynthesis pathway. Revertants EK20 (EK6) and RK13 (D4) have normal carboxysomes, which means that the restoration of the ability to grow under low CO2 coincides with the proper assembling of these structures. N5, a transport mutant due to the alteration of the gene encoding subunit 2 of NADH dehydrogenase, shows an increase in the number and size of carboxysomes and frequent bar-shaped ones.

The genomic region of rbcLS in Synechococcus sp. PCC 7942 contains genes involved in the ability to grow under low CO2 concentration and in chlorophyll biosynthesis.

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.

Physiological alterations induced by the organophosphorus insecticide trichlorfon in Anabaena PCC 7119 grown with nitrates

Abstract The addition of trichlorfon to nitrate-containing cultures of Anabaena PCC 7119 results in a decrease in the content of all the main nitrogen compounds, and an increase in the carbohydrate fraction per unit dry weight. Photoevolution of O2 per unit chlorophyll is not affected but this activity per unit dry weight is diminished due to a decrease in photosynthetic pigments. Nitrate uptake is also lowered. Cell division and morphology are altered. All these trichlorfon-induced alterations are noticeable from the first 24 hr of treatment but inhibition of growth does not occur until the fourth day.

Variation in Growth and Metabolism with Phosphorus Nutrition in two Cyanobacteria

Summary Characteristics of batch cultures of Anabaena PCC 7119 and of a wild strain of Oscillatoria (BA 010) growing either under phosphorus deficiency (P-deficiency) or with an organic source of phosphorus (P) are reported. The lowest concentration of phosphate allowing the maintenance of the batch cultures is 0.1 mM for Oscillatoria BA 010 and 0.2 mM for Anabaena PCC 7119. P-deficiency depresses growth rates, increases alkaline phosphatase activity, and alters cellular composition, the same as in other cyanobacteria. A noticeable ability to use glucose-6-phosphate (G6P) as P source has been shown for Oscillatoria BA 010, that reaches the same growth rate as control cultures, while growth of Anabaena PCC 7119 is poor. In Oscillatoria BA 010 G6P increases protein and decreases carbohydrate content. The time-course of alkaline phosphatase in G6P grown cultures shows that the initially increased levels of activity drop to control culture levels in the cyanobacterium that uses the G6P efficiently while they do not decay in the other cyanobacterium. The percentages of Oscillatoria BA 010 intracellular and cell wall bound activities are modified by the availability and source of P. Both Oscillatoria BA 010 and Anabaena PCC 7119 can be classified among the cyanobacteria that secrete the enzyme into the medium. The percentage of activity in the medium is modified by the availability and source of P in Oscillatoria BA 010 but not in Anabaena PCC 7119. The results of this work indicate that the different ability of different cyanobacteria to grow with an organic source of P depends on their higher or lower P-requirement and on their capacity to increase the alkaline phosphatase activity.

Effect of trichlorfon on N2-fixing cyanobacteriumAnabaena PCC 7119

The growth ofAnabaena PCC 7119 is affected by the organophosphorus insecticide trichlorfon, the inhibitory effect being dependent on the concentration of insecticide on the culture medium. The inhibition can be related to the unbalanced cell composition of trichlorfon-grown cells that have a decreased amount of phycobiliproteins, chlorophylls, and total proteins and an increased concentration of carbohydrates. An explanation for the lowered content in nitrogen compounds may lie in the inhibitory effect of the insecticide on N2 fixation. Cell cycle is also affected since cell volume increases, irregular divisions occur, and the number of cells per filament is shortened. However heterocysts frequency is not altered. The sensibility ofAnabaena PCC 7119 indicates that trichlorfon may affect negatively natural populations of cyanobacteria.

Alteration of dinitrogen fixation and metabolism in Cyanobacterium Anabaena PCC 7119 by phosphamidon

Abstract The effects of phosphamidon (2-chloro-2-diethylcarbamoyl-1-methylvinyl dimethyl phosphate) on carbon and nitrogen metabolism of Anabaena PCC 7119 were investigated under controlled environmental laboratory conditions. The primary action of the insecticide is dinitrogen fixation, which is negatively affected after 24 hr of treatment with 10 μg/ml. This inhibition leads to a delayed effect on cellular composition. Photosynthetic pigments, protein, nucleic acids and carbohydrates are affected from dosages of 40–60 μg/ml. Photosynthetic O2 evolution is significantly reduced after 72 hr and 50 μg/ml and is a consequence of the reduction on the photosynthetic pigments. These physiological alterations result in a growth reduction that can be noticed from 20 μg/ml (72 hr) or 300 μg/ml (48 hr). Microscopic examination of cultures treated with 300 μg/ml revealed vegetative cell swelling and heterocyst deterioration. However, heterocyst frequency was not altered. Anabaena PCC 7119 does not degrade phosphamidon.

Cell Cycle Alteration and β-1,4 Endoglucanase in Heterotrophic Chlorella vulgaris UAM 101

Summary In Chlorella vulgaris UAM 101 synthesis of copies of the DNA occurs prior to the successive series of mitoses plus cytokinesis that give rise to the autospores. Glucose promotes a cell division delay leading to the appearance of giant cells both in mixotrophic conditions as well as in photoheterotrophic and chemoheterotrophic ones. This giantism phenomenon is closely related to the increase in cell wall extensibility mediated by s-1,4 endoglucanase activity. Glucose does not interfere either with DNA synthesis or with cytokinesis but rather with mitosis. The increase in cellular size is related to the increase in cellular content of DNA. Image analysis of nuclear size indicates the presence of seven size classes of nuclei in the population of mixotrophic vegetative cells. The synthesis of these multiple copies of DNA can be related to the decrease in the nuclear-cytoplasmic ratio promoted by glucose. The giant cells only undergo mitosis when accompanied by cytokinesis and lower s-1,4 endoglucanase activities. In this case they liberate a high number of autospores. We propose that the stimulation of s-1,4 endoglucanase activity recorded is most probably due to the acidification of periplasmic space generated by the glucose uptake system. The higher activity of this enzyme in the short term and glucose availability in the long term would contribute to the regulation of cell wall synthesis and subsequent nuclear and cell division initiation.