Stimulation of Cell Division by Croton Oil in Blue-Green Bacteria

The potent tumor-promoting agent croton oil, which has been shown previously to be strongly mitogenic in mammalian cells, stimulates cell division in snake mutants of the blue-green bacterium Agmenellum quadruplicatum.     

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.     

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.     

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.     

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.     

Glycocalyx of Agmenellum quadruplicatum

The marine cyanobacterium Agmenellum quadruplicatum was shown to possess an extracellular glycocalyx similar in structure to those surrounding other bacteria from a variety of natural environments. Thin sections of cells stained with ruthenium red and frozen-etched preparations of unfixed cells indicated the glycocalyx was a network of small fibrils. The glycocalyx was present during all phases of growth, and was not degraded during nutrient limitation.     

Photoheterotrophic growth of Agmenellum quadruplicatum PR-6.

The unicellular cyanobacterium Agmenellum quadruplicatum PR-6 grows in the presence of light on agar containing 10 microM 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 1 to 30 mM glycerol. A derivative strain, PR-6G2, was tolerant of 100 mM glycerol. Photoheterotrophic growth conditions had little effect on transformation competence but did decrease the viability of single cells plated onto agar, particularly cells of the parent strain.     

Novel Mutant Impaired in Cell Division: Evidence for a Positive Regulating Factor

A novel, conditional, cell-division mutant from Agmenellum quadruplicatum strain BG1 is described. During rapid growth in dilute suspensions, cell division lags behind mass increase and the cells form filaments. These filaments spontaneously divide into unit cell lengths as the culture density increases. Other conditions that favor the accumulation of metabolic products in the medium antagonize filament formations. An 80% ethanol-water extract of dried, spent medium also restores the ability of filaments to divide into cells of unit length. Our results suggest that at least one chemical factor acting as a positive effector is involved in cell division.     

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.     

Biotransformation and toxicity of aniline and aniline derivatives in cyanobacteria

Agmenellum quadruplicatum strain PR-6 and Oscillatoria sp. strain JCM grown photoautotrophically in the presence of aniline metabolized the aromatic amine to formanilide, acetanilide and p-aminophenol. The metabolites were isolated by either thin-layer, gas-liquid or high pressure liquid chromatography and identified by comparison of their chromatographic, ultraviolet absorbance and mass spectral properties with those of authentic compounds. The toxicity of aniline derivatives towards Agmenellum quadruplicatum strain PR-6 indicated that the cyanobacterium was extremely sensitive to o-, m- and p-aminophenols, and phenylhydroxylamine.Abbreviations TLC thin layer chromatography - HPLC high pressure liquid chromatography - GC/MS gas chromatography/mass spectrometry - m/e mass to charge ratio     

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.     

Application of Flow Microflorometry to the Study of Algal Cells and Isolated Chloroplasts

The applicability of flow microfluorometry (FMF) to the studyof chlorophyll-containing cells was investigated through theuse of the blue-green alga Agmenellum quadruplicatum, the greenalgae Trebouxia, Chlorella, and Euglena spp., and isolated spinachchloroplasts. When excited by laser radiation (488 nm), algalcells emitted fluorescence with intensity positively relatedto the chloro-phyll content. The chlorophyll fluorescent signalswere used further as a differential criterion in determiningrelative size based on light scattering logic and to sort mixturesof algal cells having different chlorophyll content The FMFalso was useful in estimating nucleic acid and protein contentin completely dechlorophyUized algal cells with the use of ethidiumbromide (EB) and fluoresceinisothiocyanate (FITC), respectively.     

Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity.

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.     

Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp. israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity.

A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp. israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp. strain PCC 7002). The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene. Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product. Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.     

A survey of available nitrogen sources for the growth of the blue-green alga,Agmenellum quadruplicatum

The growth response of the marine blue-green alga, Agmenellum quadruplicatum to 60 inorganic and organic nitrogen sources was studied. These compounds were offered as sole nitrogen sources. Most amino acids, most purines, and urea were good nitrogen sources for growth.     

Isolation of a deoxycytidylate methyl transferase capable of protecting DNA uniquely against cleavage by endonuclease R.Aqu I (isoschizomer of Ava I).

A sequence-specific modification methylase (M.AquI) was isolated and purified from Agmenellum quadruplicatum (Synechococcus PCC 7002). This enzyme uniquely methylates the deoxycytidylate residue in the sequence *CYCGRG indicated by the asterisk. It was shown to protect DNA against cleavage by restriction endonucleases AvaI, SmaI and XhoI, which recognize the sequences CYCGRG, CCCGGG, and CTCGAG, respectively.     

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.     

Role of Reduced Exogenous Organic Compounds in the Physiology of the Blue-Green Bacteria (Algae): Photoheterotrophic Growth of an “Autotrophic” Blue-Green Bacterium

The unicellular blue-green bacterium Agmenellum quadruplicatum strain BG-1 was found to be capable of rapid photoheterotrophic growth but unable to grow in the dark on a variety of reduced organic substrates. The generation time on glycerol was 12 h, and on CO₂, 3 h. Glycerol carbon was converted into cellular carbon with a very high efficiency. This high efficiency of carbon conversion, the action spectrum for growth on glycerol, cell pigmentation, gas exchange measurements, and immediate ability of photoheterotrophically grown cells to evolve O₂ (upon the addition of CO₂) suggest the involvement of both photosystems I and II of photosynthesis during photoheterotrophic growth.     

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.     

Comparative ultrastructure of a filamentous mutant and the wild type ofAgmenellum quadruplicatum

Summary A filamentous variant of the marine coccoid cyanophycean alga,Agmenellum quadruplicatum has been produced after treatment of cells with the chemical mutagen, N-methyl-N-nitro-N-nitrosoguanidine (NTG). The mutation to the filamentous condition is stable and has been compared ultrastructurally with the unicellular wild type. The structural basis for the maintenance of the filamentous state is due to a failure of the homogeneously-structured transverse wall to differentiate into the stratified longitudinal wall. A manifestation of this condition is the continued synthesis of the investment membrane resulting in proliferations at each constriction. The wild type cells are nearly spherical while the filamentous mutant cells are smaller and elongate. An analysis of the structure and morphology of the nucleoplasmic regions is presented for both strains and a correlation between the morphology of these regions and the unicellular and filamentous conditions is made.