Antibiotics and apochlorosis

Twenty-four out of 38 5-nitrofuran derivatives caused bleaching ofEuglena gracilis similar to that produced by streptomycin, erythromycin and other antibiotics. Derivatives with electron-donor substituents in thep-position of the benzene nucleus attached to position 2 of benzimidazole displayed the greatest bleaching activity. The addition of hydroxylamine to a growingEuglena gracilis culture together with a 5-nitrofuran interfered with the latter's bleaching effect. The mechanism of the effect of 5-nitrofurans on theEuglena gracilis chloroplast system is thus probably identical with the mechanism of the effect of streptomycin and erythromycin. A certain correlation was observed between the bleaching effect and the antibacterial activity of the various 5-nitrofurans tested.     

Protective effect of ascorbic acid on bleaching activity of N-methyl-N′-nitro-N-nitrosoguanidine and furazolidone

Exposure of autotrophic cells ofEuglena gracilis to MNNG and furazolidone results in an irreversible loss of chloroplasts leading to a production of white mutants (permanent bleaching). Induction of the white mutants is significantly decreased by ascorbic acid, especially during the first stages of this mutation process.     

Changes of the plastid system of Euglena gracilis induced with streptomycin and dihydrostreptomycin

Summary Streptomycin-like antibiotics cause hereditary and irreversible aplastidity of Euglena gracilis by inhibiting the replication of plastids, while normal cell division is maintained.Therefore, a gradual dilution of plastids takes place in a multiplying culture. Streptomycin was found to be more effective as bleaching agent than dihydrostreptomycin. The cells of Euglena gracilis are totally deprived of plastids by streptomycin treatment after 4.5 cell divisions, while 9 cell divisions are required with dihydrostreptomycin. In addition to the inhibition of plastid replication both antibiotics bring about formation of pathological plastids, both in growing and in stationary cultures. In this latter case pathological plastids are released from cells only after further cell division has taken place.     

Isolation, culture and characterization of a new strain of Euglena gracilis

A new strain of Euglena gracilis Klebs has been isolated from a highly polluted river; it was named MAT. Strain growth in different culture media was evaluated under heterotrophic and autotrophic conditions. Total lipid, sugar, protein and chlorophyll a production were studied. Results obtained for MAT were compared with data obtained for a UTEX Culture Collection strain. Likewise, cells from both strains were bleached using streptomycin, and grown in the same media used for green samples. Both MAT and UTEX showed clear differences in their biochemical composition and growth rate depending on the media used. They also exhibited different growth patterns. E. gracilis medium proved to be the best culture environment for both strains either in autotrophic or heterotrophic conditions. Results show that basal contents of lipids, sugars, proteins and chlorophyll a vary depending on the strain, and thus values obtained for one strain do not apply to another. Moreover, strain origin may have an influence on the mechanisms of adaptation or defense developed by each sample.     

32P-Markierung der Nucleinsäuren und Bedeutung des Phosphats für den Nucleinsäure-Stoffwechsel bei Euglena gracilis

Zusammenfassung Der Einbau von ³²P-Orthophosphat in die Nucleinsäuren grüner und gebleichter Zellen von Euglena gracilis wurde untersucht. Optimale ³²P-Kurzzeit-Markierung konnte nach 4 Std Kultur der Zellen in phosphatfreiem Medium, gefolgt von einer Inkubation mit ³²P-Orthophosphat für 2 Std, erreicht werden. Die heterotrophen Zellen zeigten dabei einen etwa 5,5fach höheren Einbau als die autotrophen. Die Bedeutung von Phosphat für den Nucleinsäure-Stoffwechsel autotroph kultivierter Zellen wird anhand von Verarmungsversuchen aufgezeigt. Zunehmende Phosphatverarmung führte zunächst zum Abbau der niedermolekularen RNS, später zur Reduzierung der hochmolekularen ribosomalen RNS; die DNS wird hingegen erst relativ spät von der Verarmung betroffen. Nach 144 Std im phosphatfreien Medium erholten sich die Zellen relativ schnell, wenn sie in komplettes Nährmedium überführt wurden: nach 45 Std Erholungszeit war die Garnitur ihrer Nucleinsäuren wieder komplett. Die extrahierten Gesamtnucleinsäuren phosphatverarmter Zellen enthielten kondensierte anorganische Phosphate. Ihre Menge und Zusammensetzung änderte sich in Abhängigkeit von der Dauer der Verarmung.

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³²P-Labelling of the nucleic acids and the role of phosphate in the nucleic acid metabolism of Euglena gracilis

Summary The incorporation of ³²P _i into the nucleic acids of green and bleached cells of Euglena gracilis was investigated. Optimum short-time labelling could be achieved by subjecting the cells to phosphate starvation for 4 h followed by an incubation with ³²P _i for 2 h. In bleached cells the amount of ³²P incorporated was about 5.5 times higher than that in autotrophic cells. The role of phosphate in the nucleic acid metabolism of autotrophic cells was demonstrated by the results of starvation experiments. Under this condition the various nucleic acid components were degraded in the following order: soluble RNA, ribosomal RNA, DNA. However, even after 144 h of phosphate starvation the autotrophic cells were found to recover rather rapidly when transferred to normal growth conditions. 45 h after the transfer the nucleic acids were again present in the normal pattern. Nucleic acid preparations from phosphate starved cells contained various types of condensed inorganic phosphates. According to the length of the starvation time their amounts and compositions differed as indicated by the chromatographic behavior.

     

Protective effect of sodium selenite on ofloxacin-induced loss of chloroplast DNA inEuglena gracilis

Some xenobiotics induce permanent loss of chloroplasts inEuglena gracilis which results in the growth of white mutant colonies (bleaching effect). The effect of ofloxacin and sodium selenite on the organization of chloroplast DNA ofE. gracilis was studied by fluorescent microscopy. In the presence of ofloxacin DNA-specific staining with DAPI revealed a decrease in chloroplast DNA content and in the number of nucleoids per chloroplast. This was accompanied with a decrease of chloroplast number per cell and with the loss of stigma. Spectrophotometry of chlorophyll extract revealed changes in th ratio of chla: chlb. The presence of sodium selenite protected chloroplast DNA against the inhibitory effect of ofloxacin.     

Die Nucleinsäuren grüner und gebleichter Zellen von Euglena gracilis

Zusammenfassung Bei der Isolierung der Gesamtnucleinsäuren aus autotroph gewachsenen Zellen von Euglena gracilis und ihrer säulenchromatographischen Auftrennung an methyliertem Serumalbumin nach den Verfahren, welche für Blau- und Grünalgen optimale Ergebnisse erbrachten, konnten nur drei Komponenten der löslichen RNA und zwei der hochmolekularen RNA nachgewiesen werden. Zellaufschluß in Gegenwart von Na-Dodecylsulfat (SDS) führte auch zur Extraktion der nativen DNA, welche im Eluierungsdiagramm als zusätzliches Absorptionsmaximum erschien. Mixotroph gewachsene Zellen besaßen die gleiche Zusammensetzung an Nucleinsäuren wie autotrophe. Mittels Dichtegradienten-Zentrifugation ließen sich die Masse der DNA in einem Sediment, die der Plastiden in einer grünen Bande anreichern. Letztere enthielten je eine Komponente DNA und hochmolekulare RNA; die Komponenten der löslichen RNA, sowie die Hauptmenge der hochmolekularen RNA blieben dabei in dem Überstand, welcher bei der ersten niedertourigen Zentrifugation des Ausgangshomogents angefallen war.In permanent gebleichten Zellen (durch Streptomycin, erhöhte Temperatur) ließen sich analog die Komponenten der löslichen RNA und die DNA nachweisen, jedoch fehlte bei der hochmolekularen RNA die typische Aufteilung in die zwei Komponenten (IV und V). Die Basenzusammensetzung derselben war identisch, unabhängig von autotropher oder mixotropher Anzucht der Zellen. Auch zwischen den beiden Teilfraktionen der hochmolekularen RNA aus gebleichten Zellen bestand kein Unterschied hinsichtlich des Basenverhältnisses; ein Vergleich mit den grünen Zellen zeigte jedoch, daß der Gehalt an Cytosin höher, der an Adenin niedriger lag.

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Summary The extraction of total nucleic acids from autotrophically grown cells of Euglena gracilis and their chromatographic separation on methylated serum albumin recently successfully applied to blue-green and green algae cells yielded 3 fractions of soluble RNA and two of high molecular RNA only. Cell disruption in the presence of SDS, however, made the native DNA extractable which appeared consequently as an additional peak in the chromatographic pattern. Cells from mixotrophic cultures had an analogous set of nucleic acids compared with those from autotrophic ones. Density gradient centrifugation gave rise to a sediment containing the bulk DNA, and to a band in which the plastids had accumulated. From ther latter a fraction of DNA and high molecular RNA was isolated; the soluble RNA and the bulk high molecular RNA was associated with a supernatant resulting from an initial low speed centrifugation of the original homogenate.Cells permanently bleached (by Streptomycin, increased temperature) had the typical set of soluble RNAs and DNA; the high molecular RNA, however, was eluted in a more or less uniform peak. The base composition of the fractions IV and V of high molecular RNA was identical, independent of autotrophic or mixtrophic growth of the cells. The two partial fractions of the high molecular RNA of bleached cells were identical, too, as far as the base ratio is concerned; compared with the green cells, however, the content of cytosine tended to increase, that of adenine to decrease.

     

Furan derivatives their common molecular denominator responsible for bleaching of Euglena gracilis

Summary We studied the effect of 38 furan derivatives on the green micro-organism Euglena gracilis and on Bacillus subtilis and Escherichia coli, with the aim of finding the common molecular denominator responsible for bleaching of Euglena gracilis by furan derivatives. The results confirmed that the nitro-group in position 5 of the furan nucleus is essential to the bleaching activity of these derivatives. It was also found, however, that the substituent in position 2 of the furan nucleus was likewise important. Only 5-nitrofuran derivatives with a –CH=N–linkage in position 2 of the furan nucleus (Schiff bases) are active bleachers. Derivatives with a –CH=CH– linkage are weak bleachers and derivatives with a thiourea at this site have no bleaching activity at all. All 5-nitrofurans and some bromo- or iodo-furans were active against Bacillus subtilis, but not one of the tested derivatives was active against Escherichia coli.     

Ultraviolet-B inhibition of motility in green and dark bleachedEuglena gracilis

The motility of green and dark bleachedEuglena gracilis was studied under artificial and solar UV-B radiation. The percentage of motile cells in the population was drastically impaired after exposure to unfiltered sunlight for only a few hours. Dark bleached cells were even more affected than green organisms. The effect is caused mainly by the solar UV-B component, since filtering the sunlight by either a layer of ozone or a UV-B-absorbing filter substantially increased the survival rate. Addition ofp-quinone, a scavenger of free radicals produced in a type I photodynamic reaction, did not relieve the UV-B effects, but was cytotoxic at higher concentrations. Likewise, 1,4-diazobicyclo[2,2,2]octane and imidazole, which quench singlet oxygen (¹O₂) generated in a type II photodynamic reaction, did not prolong the survival in UV-B irradiation. D₂O, which, in contrast, prolongs the lifetime of¹O₂, is tolerated by the cells but does not aggravate the UV-B inhibition. Thus, photodynamic processes of both type I and II can be ruled out as possible mechanisms of UV-B inhibition of motility inEuglena gracilis.     

Bleaching of euglenas by antibiotics—a specific form of antagonism in actinomycetes

The author discusses little investigated antagonistic effects of certain antibiotics, manifested by the inhibition of chlorophyll synthesis inEuglena gracilis. Two main types of depigmentation were found—temporary and permanent. The oldest substrains ofEuglena gracilis var.bacillaris, strain Mainx, permanently bleached by erthromycin, streptomycin, carbomycin, spiramycin, kanamycin and viomycin, were kept in the light for at least two years without showing any signs of turning green. These antibiotics caused permanent apochlorosis, but reversible apoplastidy. The euglena cultures remained permanently heterotrophic, Picromycin, methymycin and neomycin. however, only temporarily inhibited chlorophyll synthesis. When transferred to medium not containing these antibiotics and incubated in the light, these cultures again produced chlorophyll.     

Cytological Characterization of a Giant Strain of Euglena gracilis Obtained from Dark-starved Cultures

Euglena gracilis green cells were dark-starved for four months. After this period almost the entire population died, while a few giant, viable cells appeared in the culture. The giantism was maintained after repeated subcultures in growth medium in light or dark conditions. However, the phenomenon was not permanent, and the morphological characteristics of the wild-type Euglena were gradually restored. In giant cells nuclei enlarged greatly, DNA content increased and the Golgi apparatus greatly proliferated. Chloroplasts and mitochondria increased in number and size and often presented structural modifications when compared with normal Euglena. Importantly, in the giant cells that were maintained in darkness in resting or growth conditions chloroplasts persisted as structured organelles which appeared red-fluorescent under UV illumination. Whether giantism is a phenotypic or a genotypic change is still debated. In our case, the evolution of this phenomenon, chiefly the enhanced DNA content, suggests that teratism is a multiploid mutation with the possibility of a return to the normoploid condition. Constitutive chloroplasts are present in most algae, except for a few species, among which is Euglena gracilis. The persistence of differentiated plastids in darkness in giant Euglena is considered to be a return to an ancestral condition and may, therefore, be phylogenetically important.     

Bleaching euglena gracilis with antihistamines and streptomycin-type antibiotics

Summary Kanamycin, paromomycin, and neomycin, like streptomycin, permanently bleach Euglena gracilis. This effect, along with general toxicity, is opposed by Mg, histidine or a combination of pantothenate, nicotinic acid, and threonine. Such opposition is thought to be peripheral effects centered on uptake and transport.Certain antihistamines, notably tripelennamine, methapyrilene, and pyrilamine induce permanent bleaching. Diphenylhydramine and phenindamine induced temporary bleaching. Doxylamine, antazoline, pyrathiazine, pheniramine, prophenpyridamine, and promethazine did not bleach when tested up to inhibitory concentrations.Bleaching by streptomycin+heat was additive, not synergistic.The evidence at hand for the mode of action of ultraviolet irradiation, streptomycin antibiotics, and erythromycin suggests, as a working hypothesis, that the common factor may be interference with nucleic acid metabolism; the common factor in bleaching by antibiotics may be simultaneous provision of a molecular grouping favoring uptake and transport of the active moiety, which in turn may be rare sugars interfering with ribose and desoxyribose in the photosynthetic apparatus.New antibiotics of the streptomycin family might well be screened for bleaching activity as a possible index of damage to the 8th cranial nerve, for so far the correlation is excellent for this class of antibiotic.This paper is dedicated to Professor Dr. E. G. Pringsheim on the occasion of his 80th birthday.     

Tetrahymena DNA forms a single band in alkaline density gradients

Summary DNA from nuclei ofTetrahymena pyriformis was examined by equilibrium density gradient ultracentrifugation at alkaline pH. The results indicate that the DNA has a uniform distribution of guanine plus thymine in the complementary strands and throughout the nucleotide sequence of the DNA.Abbreviations cDNA chloroplast DNA isolated fromEuglena gracilis - nDNA DNA isolated from nuclei ofTetrahymena pyriformis     

Effect of nalidixic acid on Euglena gracilis: induced loss of chloroplast deoxyribonucleic acid

Optimum conditions for bleaching of Euglena gracilis by nalidixic acid were 50 μg/ml at pH 3.8. The inhibitor did not affect cell division; however, the bleaching efficiency approached 100% after 2 generations. Within 1.5 generations of exposure to nalidixic acid the quantity of chloroplast DNA decreased at least 10-fold and was no longer detectable by equilibrium density-gradient ultracentrifugation. Replication of chloroplast deoxyribonucleic acid was completely inhibited by nalidixic acid.     

Amminoacidi Liberi in Euglena

Abstract

Free amino acids in EUGLENA. — The free amino acids present in Euglena gracilis strain Z, cultivated in the light and the dark, as well as in the strain bleached by streptomycin, have been identified by the conventional paper chromatographic method.

Variations have been observed when the photosynthetic apparatus is lost. Generally in the photosynthetic Euglena cells the pool of aminoacids reminds that of green algae, whereas bleached cells are similar to protozoa. It is possible that these variations are linked to the metabolic patways of green and bleached forms.     

Comparaison, à la lumière et à l'obscurité, des synthèses spécifiques d'ARN chez Euglena gracilis Z en cultures synchrones sur milieu lactate et étude du Chondriome

Summary Synchronization of Euglena gracilis (Z) on lactate medium is shown to be independent of illumination. The existence of a mitochondrial cycle in lightgrown as well as in dark-grown Euglena is demonstrated. When RNA synthesis is studied by pulse labeling with tritiated uracil in synchronously growing cells, a discontinuous RNA synthesis is found. Two peaks of preferential RNA synthesis in dark-grown cells and three peaks in light-grown cells are seen; the significance of the third peak of RNA synthesis in light-grown Euglena is discussed.     

EFFECTS OF DARKNESS AND OF STREPTOMYCIN ON THE FINE STRUCTURE OF EUGLENA GRACILIS

Siegesmund , Kenneth A., Walter G. Rosen , and Stanley R. Gawlik . (Marquette (J., Milwaukee, Wis.) Effects of darkness and of streptomycin on the fine structure of Euglena gracilis. Amer. Jour. Bot. 49 (2) : 137–145. Illus. 1962.—Dark-grown Euglena gracilis cells, transferred from streptomycin (SM)-containing medium to SM-free medium 5 days before transfer to light, turn green normally, indicating that proplastids are unaffected by SM. SM-bleached cells, grown in light, contain numerous bodies composed of concentric lamellae (CL bodies). These differ from chloroplasts in that their lamellae lack electron-dense dots, are not coalesced, and they lack a 3-layered limiting membrane and pyrenoids. CL bodies are absent from dark-grown normal and dark-grown SM-bleached cells, as well as from light-grown normal cells. It is suggested that CL bodies result from a derangement of chloroplast synthesis caused by SM blockage of chlorophyll synthesis.     

Nitrofuran-Reducing Enzymes of Euglena*

SYNOPSIS. Euglena gracilis strain Z, green, dark-grown, and “bleached”with N-methyl-N-nitro-N-nitrosoguanidine, was found to contain 2 soluble enzymes which reduce nitrofurans. A small amount of activity was demonstrated also in a particulate fraction of sonic extracts, but none in isolated chloroplasts. The reduction of 5 nitrofurans, having widely differing bleaching activities, by each of the 2 enzymes was examined.     

Glycine cleavage and generation of one-carbon units in Euglena gracilis

Euglena gracilis Klebs (strain Z) was maintained in division synchronized autotrophic culture, receiving either air (low CO₂) or 5% CO₂ in     

Mutagenic effects of streptomycin in chlamydomonas

Summary Growth of a green streptomycin-resistant strain of Chlamydomonas reinhardi on a sub-lethal concentration of streptomycin on agar led to the appearance of yellow mutant cells in almost every colony. The time of appearance of the mutants varied greatly among the 9 isolates studied, each of which was selected as a single colony after repeated cloning of the parental strain. 2 isolates gave rise to colonies which responded rapidly to streptomycin (class I), 2 isolates produced yellow sub-clones as papillae only after formation of normal green colonies (class II), and 2 isolates produced stable yellow sub-clones only after a second subculture on streptomycin-agar (class III). 3 isolates were mixtures of classes II and III.The evidence that these yellow mutants arose under the mutagenic action of streptomycin is discussed in relation to the alternative possibility of their selection by the drug from a pool of pre-existing mutants. The physiological and genetic effects of streptomycin upon chlorophyll formation in Chlamydomonas are compared with reported effects of the drug upon the green flagellate, Euglena gracilis.Dedicated with appreciation and affection to Professor Dr. E. G. Pringsheim on the occasion of his 80th birthday.