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The temperature at which Prototheca spp. were grown determined their response to freezing to -196 degrees C and subsequent thawing. Cells cultured at 35 degrees C were the most sensitive to freezing injury; at lower growth temperatures, resistance to freezing damage was seen. At all culture temperatures examined, the freezing tolerance varied with the age of the culture.
Prototheca wickerhamii was treated in vitro with 11 different herbicides and plant hormones. Growth was inhibited by indolyl-3-acetic acid, indolyl-3-butyric acid and indolyl-3-propionic acid at 400 mug per ml. Coconut milk was stimulatory.
The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a(3). Cytochromes a and a(3) could be resolved spectrally in the alpha-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a(3) complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a(3), and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).
Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a(3). Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The alpha-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a(3) was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.
Membrane preparations from the non-photosynthetic alga Prototheca zopfii incorporate glucose from UDP-[3H]glucose into the trichloroacetic-acid-insoluble fraction and the polysaccharides insoluble in hot alkali. Time course and pulse-chase experiments indicate that the acid-insoluble fraction was a precursor of the alkali-insoluble fraction. Isolation of 3H-labeled membrane or soluble fraction showed that only membrane fractions were able to transfer radioactivity into polysaccharides. Treatment of glucosylated membranes with trypsin or cellulase only partially affect their transfer ability, indicating that the precursor was internalized in vesicles. Analysis of the in vitro synthesized polysaccharides by enzymatic and acid hydrolysis showed that glucose and cellobiose were present as radioactive sugars. Permethylation of the polysaccharide indicates that 80% of the glucose was beta-1,4-bonded with 20% in beta-1,3-linkages. This polysaccharide was found to be identical with the cell-wall beta-glucan obtained in vivo [Rivas, L.A. & Pont Lezica, R. (1978) Planta (Berl.) 165, 348-353].
When Prototheca zopfii cells were pulse-labeled with 14C-containing amino acids and homogenized, 14C-labeled membranes were obtained. In vitro incubations with the previously labeled membranes and UDP-[3H]Glc produced a trichloroacetic-acid-insoluble fraction having both isotopes. A double-labeled glucoprotein was isolated and characterized. It has a relative molecular mass of 28,000-30,000 and a carbohydrate content of 10%. The oligosaccharide chain is linked to the protein through an O-glycosidic bond between hydroxyproline and glucose. The oligosaccharide has a polymerization degree ranging over 10-20 hexose units. Glucose is the only monosaccharide found; most of the glucose residues are beta-1,4-linked (90%) but some are beta-1,3-linked (10%).
Prototheca wickerhamii is a yeastlike organism that resembles the green alga Chlorella. Nuclear division in coordination with cytoplasmic cleavage gives rise to uninucleate cytoplasmic segments, each of which acquires a delicate cell wall and develops into an autospore. The autospores in this species are spherical; but in a variant that presumably arose as a result of spontaneous mutation, the cytoplasmic cleavage is irregular, and the resultant autospores are ovoid to bacillary. When these variant autospores grow, they swell and round up before the nuclear division begins, producing spherical cells like those seen in wild-type cultures. In view of the fact that species concept in the genus is based on size and shape of cells, the variation limits in these morphological characteristics have significant bearing on species classification.
A new strain of Prototheca zopfii Krüger was grown on acetate or on pure n-alkanes. A maximum acetate-supported exponential growth of 12 divisions day occurred at pH 5 and 30 degrees C. At 25 degrees C, growth on n-alkanes was almost as fast, but no growth occurred at 30 degrees C. After 4 days at 25 degrees C, 34 to 45% of the n-alkanes had been removed, whereas at 21 degrees C and slower growth, utilization was twofold greater after 15 days. Rates of growth and utilization increased markedly after a point of sudden emulsification.
A medium was devised which permitted the selective isolation of Prototheca spp. from nature.
Prototheca zopfii is an achlorophyllous alga which degrades oil. It has been found to degrade 10 and 40% of a motor oil and crude oil, respectively, when tested under appropriate conditions. Degradation of the crude oil observed in this study compares well with the amount of degradation accomplished by bacteria. P. zopfii was found to degrade a greater percentage of the aromatic hydrocarbons in motor oil than of the saturated hydrocarbons and a greater percentage of saturated hydrocarbons in crude oil than of aromatic hydrocarbons. Resins and asphaltens were produced during degradation of motor oil, whereas these fractions in crude oil were degraded. P. zopfii did not demonstrate preferential utilization of lower homologues of cycloalkanes and aromatics as has been observed with bacteria.
Bacteria and Yeasts are able to induce, when inoculated into laboratory rodents, a general stimulation of defences by an immune process. We could estimate that unicellular algae would induce the same phenomenon because the chemical compounds of their walls are related to those of the precedings microorganisms specially to bacteria. Indeed, two Chlorellaceae, Prototheca segbwema and Chlorella pyrenoidosa are respectively protecting 78% and 82% CH3 mice against the sarcoma BP8 grafting.
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The mastitis caused by P. zopfii alga is a disease of high-producing, machine-milked dairy cows. It occurs worldwide in tropical and temperate climatic areas, and mostly appears sporadically in a therapy-resistant form. However, in poorly managed dairy herds it may be endemic, causing serious economic losses as a result of decreased milk quality and quantity and culling of infected animals. The biological properties of this pathogenic alga, the laboratory methods available for its isolation and identification, the pathological and clinical features of this form of mastitis, and the principles of its control are reviewed in this paper.