Action of Nalidixic Acid on Chloroplast Replication in Euglena gracilis

The role of light in nalidixic acid bleaching of Euglena gracilis var. bacillaris was investigated. The kinetics of loss of the chloroplast-associated DNA and the sensitivity of chloroplast replication to ultraviolet light was followed during treatment with nalidixic acid. By using the mutant P₄ZUL, and 3-(3,4-dichlorophenyl)-, 1-dimethylurea, it was demonstrated that the requirement for light was a functioning photosynthetic electron transport system. Ultracentifugal analysis showed a substantial decrease in chloroplast-associated DNA after 6 hours of treatment with nalidixic acid. Ultraviolet target analysis revealed that the number of chloroplast genomes per cell had been reduced. The possible role of light and implications of the reduction in chloroplast genomes for chloroplast replication are discussed.     

Induced Changes in Chloroplast Protein Accumulation during Heat Bleaching in Euglena gracilis

When growing cultures of light-grown Euglena gracilis Z are exposed to slightly elevated temperatures (33°C) there is a time-dependent decrease in chlorophyll (bleaching) and a gradual transformation of chloroplasts into rudimentary plastids. A study was undertaken whose primary objective was to document major changes in polypeptide composition in the stroma and in thylakoids of cells that have been exposed to the bleaching temperature for up to 57 hours. A novel polypeptide of about 60,000 to 63,000 M_r whose function is presently unknown, accumulates in the stroma and in thylakoids in response to growth at the bleaching temperature. The levels of the large and small subunit of ribuolosebisphosphate carboxylase, on the other hand, decrease to very low levels at about 33 hours and remain very low for the duration of the temperature treatment. Of two polypeptides associated with the light-harvesting chlorophyll-protein complex of photosystem II (28,000 and 24,500 M_r) only the level of the smaller polypeptide decreases at the elevated temperature. The levels of 28,000 M_r species remain virtually unchanged throughout the temperature treatment period. Changes in chloroplast polypeptide composition were also studied in cells that were allowed to recover at room temperature from an initial treatment at 33°C. Bleaching Euglena could provide a useful tool for studying the interaction between the nucleus and chloroplast genetic system that govern the development and maintenance of this vital organelle to plants.     

Studies on polyamine biosynthesis in Euglena gracilis.

Euglene gracilis (strain Z) was found to contain five polyamines which could be separated by high-pressure cation-exchange chromatography. 1,3-Diaminopropane, putrescine, norspermidine (N-(3-aminopropyl)-1,3-diaminopropane), spermidine and norspermine (N,N'-bis(aminopropyl)-1,3-diaminopropane) were identified. Biosynthesis of putrescine in E. gracilis proceeds through decarboxylation of L-ornithine, no arginine decarboxylase (EC 4.1.1.19) activity could be detected. The properties of the enzymes ornithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50) in this alga were found to be similar to those of the enzymes isolated from animal tissues or yeast cells. A bioxynthetic scheme is proposed which relates the different polyamines occurring in E. gracilis.     

Action of Myxin on the Chloroplast System of Euglena gracilis

SYNOPSIS. Myxin (1-hydroxy-6-methoxy-phenazine-5,10-dioxide), a wide spectrum antibiotic, inhibits chloroplast replication in Euglena gracilis strain Z at concentrations which have no effect upon growth or survival. Myxin also inhibits the synthesis of chlorophyll when etiolated Euglena are illuminated in resting medium. By analogy with its action on bacteria, it is suggested that myxin may cause selective inhibition of chloroplast nucleic acid synthesis.     

Effect of Nitrofurans on the Chloroplast System of Euglena gracilis

SYNOPSIS. Twelve derivatives of 5-nitrofuran were tested on Euglena grarzlis . All rendered the organism permanently apoplastidic and, at somewhat higher concentrations, killed. The furan analogues of 3 of these compounds had no effect on the chloroplast system and were less toxic than the nitrcfurans. Low concentrations of nitrofurantoin and nitroiuraldehyde inhibited formation of chlorophyll when etiolated cells irere illuminated.
Exposure of euglena to low concentrations of these agents for about 2 generation times. followed by plating on drug-free mediurn. resulted in a high proportion of bleached colonies. It is theretore concluded that the nitrofurans induce apotplastidy by causing permanent damage to the chloroplast system rather than by inhibiting its replication temporarily. Since one of the nitrofurans which was found to bleach euglena, NFT–3-amino-6[-2-(5-nitro-2-furyl) vinyl]-1,2,4-triazine–is known to cause specific inhibition of DNA synthesis in bacteria, nitrofurans may perhaps bleach euglena through selective damage to chloroplast-DNA or to the DNA-synthesizing system of the chloroplast.     

Influence of Culture pH on Chloroplast Structure in Euglena gracilis

Chloroplasts of Euglena gracilis grown with phototrophic nutrition at pH 3.0 were compact, while those in cells grown at pH 8.1 were swollen with widely separated lamellae.     

Studies of Chloroplast Development in Euglena: XIII. Variation of Ultraviolet Sensitivity with Extent of Chloroplast Development

Ultraviolet (UV) inactivation of green colony-forming ability of several different types of Euglena gracilis var. bacillaris was studied. The observed target numbers are not widely different, while the doses required to produce a single inactivation event (D(o)) vary with the type of cell used. In dark-grown cells adapting to the light in resting medium and in an X-ray-induced mutant, D(o) is proportional to the chlorophyll content of the cells. However, in hyperdeveloped cells which contain abnormally high amounts of chlorophyll, the correlation does not hold, suggesting that it is not chlorophyll per se which is responsible for the differences observed. D(o)'s of colony-forming ability (viability) of light-grown and dark-grown cells are found to differ by the same factor as those of green colony-forming ability. Stationary phase and exponential phase cells show a small difference in D(o) with no obvious difference in target multiplicity. The multiplicity of the various target curves has been re-evaluated by computer and found to be between 30 and 40.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

Studies in Carotenogenesis. Some Observations on Carotenoid Synthesis in Two Varieties of Euglena gracilis.

Euglena gracilis v. bacillaris and E. gracilis v. fuscopunctata produce the same three carotenoids, β-carotene, lutein and neoxanthin in approximately the same relative amounts. Lutein is the major pigment and β-carotene represents about 10–15% of the total. The concentration of carotenoids in E. gracilis v. bacillaris is very high, reaching 700 mg./100 g. dry weight.
Streptomycin (0.02%, w/v) and darkness reduced growth of E. gracilis v. bacillaris by about 50% and carotenoid synthesis by about twenty times. Diphenylamine (1/70,000) reduced growth and carotenoid synthesis equally (about 50%). In no case was the synthesis of more saturated polyenes (the phytofluene series) stimulated.
The nature of the eye spot pigment is discussed.     

Action of Nitrofuran Derivatives on the Chloroplast System of Euglena gracilis: Effect of Light

SYNOPSIS. When cultures of Euglena are exposed to nitro-furantoin in the light, there is extensive bleaching and, at higher concentrations, killing. In the dark the same concentrations have essentially no effect. The enhanced activity in light is a consequence of the rapid photolysis of nitrofurantoin to yield 5-nitro-2-furaldehyde which is, in turn, destroyed by light. Nitrofuraldehyde and several other nitrofuran derivatives bleach at low concentrations in the dark.
Of 14 nitrofuran derivatives tested, only 4 Schiff's bases which were readily decomposed to nitrofuraldehyde in light were more active in the light than in the dark.     

Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide.

Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellate Euglena gracilis were purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol of E. gracilis were cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor-Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase of E. gracilis showed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme of E. gracilis did not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi.