Effects of Iron Starvation on the Ultrastructure of the Cyanobacterium Agmenellum quadruplicatum

The effects of iron starvation on the ultrastructure of the unicellular cyanobacterium Agmenellum quadruplicatum were studied by using thin sectioning and transmission electron microscopy. Intracellular polysaccharide began to accumulate at the onset of iron limitation. This was followed by degradation of ribosomes and (later) degradation of the thylakoid membranes, both of which were virtually absent by 200 h. The thylakoids underwent structural modifications and rearrangements before they actually began to break down. Iron starvation did not appear to affect carboxysomes or the extracellular glyocalyx. On the other hand, polyphosphate bodies may have been partially degraded, and an electrontransparent gap eventually appeared between the cell wall and the cytoplasmic membrane. All of these changes were reversed when iron was added back to 200-h starved cultures. The sequence of ultrastructural changes observed during iron starvation clearly differed from those previously reported to occur during nitrogen, phosphorous, or carbon limitation.     

Effects of Iron Starvation on the Physiology of the Cyanobacterium Agmenellum quadruplicatum

The effects of iron starvation on the growth and physiology of the unicellular cyanobacterium Agmenellum quadruplicatum were studied. Uptake of iron from the medium did not occur at a constant rate. The majority of the iron was removed at two different times, when the cells were initially inoculated into the medium and after the cultures had become quite dense and had stopped growing. Iron became limiting for growth 16 h after transfer to an iron-deficient medium, but cultures retained full viability for at least 212 h. Once iron became limiting, c-phycocyanin and chlorophyll a were degraded concurrently. This was followed by an accumulation of intracellular glucose in place of the c-phycocyanin. Nitrate and nitrite reductase activities were elevated through 50 h, after which they decreased steadily. The photosynthetic unit size also increased through 50 h and then decreased. Once iron was restored to the culture medium, growth resumed. The intracellular pigment levels increased rapidly as the glucose level diminished.     

Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum.

Chromosomal transformation of Agmenellum quadruplicatum PR-6 (= Synechococcus sp. strain 7002) was characterized for phenotypic expression, for exposure time to DNA, and for dependence on DNA concentration with regard to Rifr donor DNA. Exponentially growing cells of PR-6 were competent for chromosomal transformation. Competence decreased in cells in the stationary phase of growth or in cells deprived of a nitrogen source. Dark incubation of cells before exposure to donor DNA also decreased competence. Homologous Rifr and Strr DNA and heterologous Escherichia coli W3110 DNA were used in DNA-DNA competition studies, which clearly showed that DNA binding by PR-6 was nonspecific. DNA binding and uptake by PR-6 exhibited single-hit kinetics. Single-stranded DNA failed to transform competent cells of PR-6, and DNA eclipse was not observed, suggesting that double-stranded DNA was the substrate for the binding and uptake reactions during the transformation of PR-6. A significant improvement in transformation frequency was achieved by increasing the nitrate content of the culture medium and by lowering the temperature at which cells were exposed to donor DNA from 39 degrees C (the optimal temperature for growth) to 30 degrees C.     

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

Biosynthesis of delta-Aminolevulinic Acid from Glutamate in Agmenellum quadruplicatum

δ-Aminolevulinic acid accumulated in the culture medium when Agmenellum quadruplicatum strain PR-6 was incubated in the presence of levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydratase, and specifically labeled glutamate and glycine. The δ-aminolevulinic acid was purified using Dowex 50W-X8 and cleaved by periodate to yield succinic acid and formaldehyde. The distribution of radioactivity in the two fragments suggested that in blue-green algae the carbon skeleton of δ-aminolevulinic acid is derived directly from glutamate. However the possibility of the pathway of δ-aminolevulinic acid synthesis, from glycine and succinyl-coenzyme A also functioning in blue-green algae was not eliminated as uptake of glycine was minimal.     

Ribonucleotide reductase activity in ether-treated cells of Agmenellum quadruplicatum.

Ribonucleotide (cytidine 5'-diphosphate) reductase activity can be detected in situ in cells of the blue-green alga Agmenellum quadruplicatum, strain PR-6, after the cells are made permeable by treatment with ether. The Agmenellum reductase resembles the enzyme from Escherichia coli.     

Metabolism of phenanthrene by the marine cyanobacterium Agmenellum quadruplicatum PR-6.

Under photoautotrophic growth conditions, the marine cyanobacterium Agmenellum quadruplicatum PR-6 metabolized phenanthrene to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) and 1-methoxyphenanthrene as the major ethyl acetate-extractable metabolites. Small amounts of phenanthrols were also formed. The metabolites were purified by high-pressure liquid chromatography and identified from their UV, infrared, mass, and proton magnetic resonance spectral properties. A. quadruplicatum PR-6 formed phenanthrene trans-9,10-dihydrodiol with a 22% enantiomeric excess of the (-)-9S,10S-enantiomer. Incorporation experiments with 18O2 showed that one atom of oxygen from O2 was incorporated into the dihydrodiol. Toxicity studies, using an algal lawn bioassay, indicated that 9-phenanthrol and 9,10-phenanthrenequinone inhibit the growth of A. quadruplicatum PR-6.     

Metabolism of phenanthrene by the marine cyanobacterium Agmenellum quadruplicatum PR-6.

Under photoautotrophic growth conditions, the marine cyanobacterium Agmenellum quadruplicatum PR-6 metabolized phenanthrene to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) and 1-methoxyphenanthrene as the major ethyl acetate-extractable metabolites. Small amounts of phenanthrols were also formed. The metabolites were purified by high-pressure liquid chromatography and identified from their UV, infrared, mass, and proton magnetic resonance spectral properties. A. quadruplicatum PR-6 formed phenanthrene trans-9,10-dihydrodiol with a 22% enantiomeric excess of the (-)-9S,10S-enantiomer. Incorporation experiments with 18O2 showed that one atom of oxygen from O2 was incorporated into the dihydrodiol. Toxicity studies, using an algal lawn bioassay, indicated that 9-phenanthrol and 9,10-phenanthrenequinone inhibit the growth of A. quadruplicatum PR-6.     

Isolation of a small-cell mutant in the blue-green bacterium Agmenellum quadruplicatum.

A new type of high-temperature conditional cell division mutant has been isolated in Agmenellum quadruplicatum strain BG1 in which the process of cell division is uncoupled from that of growth at 39 C. This mutant produces abnormally small cells under conditions of nutrient limitation and forms multinucleoid filaments under normal growth conditions.     

Effect of levulinic acid on pigment biosynthesis in Agmenellum quadruplicatum.

When levulinic acid was added to a growing culture of the cyanobacterium (blue-green alga) Agmenellum quadruplicatum PR-6, delta-aminoelevulinic acid accumulated in the medium and chlorophyll a synthesis and cell growth were inhibited, but there was a small amount of c-phycocyanin synthesis. The amount of delta-aminolevulinic acid produced in the treated culture did not fully account for the amount of pigment synthesized in the untreated control. Levulinic acid and either sodium nitrate or ammonium chloride were added to nitrogen-starved cultures of PR-6, and delta-aminolevulinic acid production and chlorophyll a and c-phycocyanin content were monitored. When ammonium chloride was added as a nitrogen source after nitrogen starvation, the cells recovered more rapidly than when sodium nitrate was added as a nitrogen source. In cultures recovering from nitrogen starvation, synthesis of c-phycocyanin occurred before synthesis of chlorophyll a.     

Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6

The 130-kilodalton mosquito larvicidal gene, cloned from Bacillus thuringiensis var. israelensis, was introduced into the cyanobacterium Agmenellum quadruplicatum PR-6 by plasmid transformation. Transformed cells synthesized 130-kilodalton delta-endotoxin protein and showed mosquito larvicidal activity. Results demonstrate a potential use of a cyanobacterium for biological control of mosquitoes.     

Characteristics of Nitrate Reduction in a Mutant of the Blue-Green Alga Agmenellum quadruplicatum

Characteristics of nitrate reduction in terms of nitrite production in an N-methyl-N′-nitro-N-nitrosoguanidine-induced mutant of the blue-green alga Agmenellum quadruplicatum are described. Following induction of nitrate reduction a linear rate of nitrite production proportional to cell concentration was observed. Rate of nitrite production and growth rate showed similar responses to pH, temperature, and light intensity. If required, only trace amounts of carbon dioxide were necessary for nitrite production. Atmospheres of oxygen or nitrogen inhibited production of nitrite. In addition, a low but constant rate of nitrite production was observed in the dark. Nitrite production by mutant AQ-6 was studied in terms of photosynthesis. As nitrite production proceeded, rate of photosynthesis declined. Ultraviolet irradiation and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea poisoning did not prevent nitrite production. The action spectrum of nitrite production was chlorophyll a-like.     

Carbon-13 NMR analysis of cyclic peptide alkaloids

The chemical shifts of the carbons of the peptide alkaloids discarine-A, discarine-B, lasiodine-A, lasiodine-B, pandamine, pandaminine and hymenocardine have been assigned.     

The effects of nitrogen limitation on the ultrastructure of the cyanobacterium Agmenellum quadruplicatum

The effects of nitrogen limitation on the ultrastructure of the unicellular cyanobacterium, Agmenellum quadruplicatum, were studied by thin sectioning transmission electron microscopy. Nitrogen became limiting for growth 14–15 h after transfer to nitrogen-limiting medium, but cultures retained full viability for at least 45 h. The c-phycocyanin: chlorophyll a ratio and cellular nitrogen content of the culture dropped rapidly after 14–15 h, as a progressive deterioration of major cell structures took place. Phycobilisomes were degraded first, followed by ribosomes and, then, thylakoid membranes. These structures were virtually depleted from the cells within 26 h. Intracellular polysaccharide accumulated in place of the normal cell structures throughout this period. Nitrogen limitation did not affect polyphosphate bodies, carboxysomes, lipid granules, the cell envelope, or the extra-cellular glycocalyx. All of the ultrastructural changes resulting from nitrogen limitation were reversed upon addition of nitrate to a starved culture. Most cell structures were restored within 3 h, and restoration was complete within 9 h.     

Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6.

The 130-kilodalton mosquito larvicidal gene, cloned from Bacillus thuringiensis var. israelensis, was introduced into the cyanobacterium Agmenellum quadruplicatum PR-6 by plasmid transformation. Transformed cells synthesized 130-kilodalton delta-endotoxin protein and showed mosquito larvicidal activity. Results demonstrate a potential use of a cyanobacterium for biological control of mosquitoes.     

Selective Inhibition of Deoxyribonucleic Acid Synthesis by 2-Deoxyadenosine in the Blue-Green Bacterium Agmenellum quadruplicatum

Concentrations of deoxyadenosine which have little effect on net ribonucleic acid (RNA) synthesis or on increase in cell mass selectively inhibit deoxyribonucleic acid (DNA) synthesis in Agmenellum quadruplicatum. Exogenously supplied deoxyadenosine, at concentrations above 10 mug/ml, stimulates DNA degradation. These results are correlated with a rapid loss in viability. Over a narrow concentration range (6-15 mug/ml), deoxyadenosine impairs the division process and induces the formation of elongated cells. Low exogenous concentrations of deoxyadenosine are readily incorporated into both DNA and RNA, with the major portion as DNA.     

Accumulation of Cyanophycin Granules as a Result of Phosphate Limitation in Agmenellum quadruplicatum

Phosphate-limited growth of the blue-green alga Agmenellum quadruplicatum resulted in the accumulation of cyanophycin granule polypeptide (CGP), which is a 1:1 co-polymer of aspartic acid and arginine. The progressive accumulation of CGP began after depletion of phosphate from the medium. CGP increased in concentration much faster than the increase in cell number. Electron microscopy indicated that both the number of cyanophycin granules per cell section and the diameter of each granule increased as phosphate starvation progressed. A marked decrease in the electron density of the inter-thylakoidal areas took place concurrently with the accumulation of CGP. At the same time a progessive decrease in the pigment concentration of cells and in the rate of nitrate uptake was observed. Thirty-two hours after phosphate depletion from the medium up to 28% of total cellular nitrogen was found in CGP.     

Carbon-13 NMR studies of 13CO binding to human hemoglobin

In the ¹³C NMR spectrum of hemoglobin A carbonylated with ¹³CO, separate resonances can be distinguished at 207.04 ppm and 206.60 ppm (with respect to the ¹³C resonance of external tetramethyl-silane) for ¹³Co bound to the α and β chains of the hemoglobin tetramer. A study of the ¹³Co derivatives of the isolated α and β chains, and of the abnormal hemoglobin M_IWATE which contains α chains which are in the met [Fe(III)] form and do not bind CO, has permitted an assignment of the high field (206.60 ppm) resonance to the β chain ¹³CO and the low field one to the α chain ¹³CO. The identification of these ¹³Co resonances permits a study of the differences in the chemistry of the α and β heme units in intact hemoglobin. Some results on the differences in the redox behavior of these chains are included.