Ochromonas Malhamensis Research Articles

GRAZING AND GROWTH OF THE MIXOTROPHIC CHRYSOMONAD POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) FEEDING ON ALGAE

The growth and grazing characteristics of Poterioochromonas malhamensis (Pringsheim) Peterfi (= Ochromonas malhamensis Pringsheim) (ca. 8 μm) feeding on phytoplankton, including the cyanobacteria Synechococcus sp. (ca. 2 μm) and Microcystis viridis (A. Brown) Lemmermann (ca. 6 μm) and the green alga Chlorella pyrenoidosa Chick (ca. 13 μm), were investigated in laboratory experiments involving the following treatments: (1) light without added algal prey (autotrophy), (2) light with added algal prey (mixotrophy), and (3) dark with added algal prey (phagotrophy). There were significantly higher cell numbers under mixotrophic and phagotrophic growth than under autotrophic growth. With phytoplankton as food, growth rates under both mixotrophy and phagotrophy were about two or three times higher than those under autotrophy, indicating that the algal diets were readily able to support the population growth of P. malhamensis. There were no significant differences in growth rate between mixotrophic and phagotrophic cultures during exponential growth. The ingestion rate of P. malhamensis with algal prey was also similar under both continuous light and dark. Poterioochromonas malhamensis ingested on average 0.27 M. viridis cells·flagellate−1·h−1 and 0.18 C. pyrenoidosa cells·flagellate−1·h−1 in continuous light and 0.25 M. viridis cells·flagellate−1·h−1 and 0.18 C. pyrenoidosa cells·flagellate−1·h−1 in continuous dark during exponential growth. The results showed that light had no effect on the growth and ingestion rates of P. malhamensis for phagotrophy during exponential growth. However, phagotrophic populations of P. malhamensis were incapable of growth in continuous darkness for longer than 5 days. Populations of P. malhamensis showed no increase when prey was added again after 4 days in continuous darkness, indicating that light is necessary for sustained phagotrophic growth of P. malhamensis. The study suggests that P. malhamensis, which has strong tolerance for light, is light dependent for phagotrophy.

Biotransformation of benzo[ a]pyrene and other polycyclic aromatic hydrocarbons and heterocyclic analogs by several green algae and other algal species under gold and white light

This laboratory has shown that the metabolism of benzo[ a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon (PAH), by a freshwater green alga, Selenastrum capricornutum, under gold light proceeds through a dioxygenase pathway with subsequent conjugation and excretion. This study was undertaken to determine: (1) the effects of different light sources on the enzymatic or photochemical processes involved in the biotransformation of BaP over a dose range of 5–1200 mg/1; (2) the phototoxicity of carcinogenic PAHs and mutagenic quinones to a green alga; (3) the ability of other algal systems to metabolize BaP. Cultures were exposed to different doses of BaP for 2 days at 23 °C under gold, white or UV-A fluorescent lights on a diurnal cycle of 16 h light, 8 h dark. Under gold light, metabolites of BaP produced by Selenastrum capricornutum were the dihydrodiols of which the 11,12-dihydrodiol was the major metabolite. Under white light, at low doses, the major metabolite was the 9,10-dihydrodiol. With increasing dose, the ratio of dihydrodiols to quinones decreased to less than two. With increasing light energy output, from gold to white to UV-A in the PAH absorbing region, BaP quinone production increased. Of other carcinogenic PAHs studied, only 7H-dibenz[ c, g]carbazole was as phototoxic as BaP while 7,12-dimethylbenz [ a]anthracene, dibenz[ a, j]acridine and non-carcinogenic PAHs, anthracene and pyrene, were not phototoxic. The 3,6-quinone of BaP was found to be highly phototoxic while quinones that included menadione, danthron, phenanthrene-quinone and hydroquinone were not. The data suggest that the phototoxicity of BaP is due to photochemical production of quinones; the 3,6-quinone of BaP is phototoxic and is probably the result of the production of short lived cyclic reactive intermediates by the interaction of light with the quinone. Lastly, only the green algae, Selenastrum capricornutum, Scenedesmus acutus and Ankistrodesmus braunii almost completely metabolized BaP to dihydrodiols. The green alga Chlamydomonas reinhardtii, the yellow alga Ochromonas malhamensis, the blue green algae Anabaena flosaquae and euglenoid Euglena gracilis did not metabolize BaP to any extent. The data indicate that algae are important in their ability to degrade PAHs but the degradation is dependent on the dose of light energy emitted and absorbed, the dose of PAHs to which the algae are exposed, the phototoxicity of PAHs and their metabolite(s) and the species and strain of algae involved. All of these factors will be important in assessing the degradation and detoxification pathways of recalcitrant PAHs by algae.

Cell inactivation in the presence of sparging and mechanical agitation

Microbial cells are more readily rendered nonviable by the combined action of air sparging and mechanical agitation than by either action along. A. bubble breakup/coalescence model that incorporates the cell-bubble encounter rate, bubble breakup rate, and death probability is proposed to describe cell inactivation in the presence of bubbles maintained through the joint action of agitation and air, which is continually fed into the impeller stream region via passive vortex entrainment from the surface above or via active sparging from below. Experimental results obtained from a fragile algal (Ochromonas malhamensis) culture are consistent with the model prediction. In particular, the specific cell death rate is linearly related to the specific bubble interfacial surface area. It is shown that cells exhibit sparging-sensitive characteristics when agitation is mild, but become sensitive to surface vortexing when agitation turns vigorous enough to introduce air entrainment. Experimental data obtained from different stirrer sizes are in good agreement with the model.

Oxygen mass transfer enhancement via fermentor headspace pressurization.

Bioreactor headspace pressurization represents an excellent means of enhancing oxygen mass transfer to a culture. This method is particularly effective in situations where stirring or vigorous aeration is difficult. Because it in itself introduces no undesirable hydrodynamic force, the proposed method is also attractive for cells susceptible to agitation and sparging. Experiments were first conducted in an ideal fermentor by sparging air into a sulfite solution free from extraneous microbial effects. An increased oxygen mass transfer rate resulting from pressurization led to a superior cell growth rate and a higher maximum cell density in both of the microbial systems studied: a bacterial (Escherichia coli) culture up to 2.72 bar and a fragile algal (Ochromonas malhamensis) culture with pressure programming. Applying pressurization increased the maximum dry cell weight from 1.47 g/L to 1.77 g/L in the E. coli culture and increased the maximum viable cell density from 4 x 10(7) cells/mL to 10(8) cells/mL in the algal culture. An additional advantage is that formation of undesirable products under oxygen limitation, e.g., acetic acid in the E. coli culture, can be suppressed. A significant (over 250%) improvement in the oxygen transfer rate can be achieved with existing fermentors with little modification as they are already designed to withstand reasonable pressure from autoclaving. This method is simple, clean, inexpensive, and easily implemented, and it can be applied alongside other existing methods of oxygen mass transfer enhancement.

Biosynthesis of poriferasterol in Ochromonas malhamensis: 13C NMR assignment of the isopropyl methyl groups of 2-methylpentan-3-ol

2-Methylpentan-3-ol stereospecifically labelled with deuterium on one of the isopropyl methyl groups has been synthesized, thus allowing the unequivocal assignment of their 13C NMR resonances. 13C NMR assignments for these groups proposed earlier are shown to be incorrect. Involvement in the biosynthesis of the 24β-ethylsterol poriferasterol in the crysophyte Ochromonas malhamensis is discussed.

Conversion of 5-S-Ethyl-5-thio-D-ribose to Ethionine in Klebsiella pneumoniae: Basis for the selective toxicity of 5-S-ethyl-5-thio-D-ribose

5-S-Ethyl-5-thio-D-ribose (ethylthioribose) exhibits antiprotozoal activity against Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis, but is nontoxic to cultured human and murine bone marrow cells (Riscoe, M. K., Ferro, A. J., and Fitchen, J. H. (1988) Antimicrob. Agents Chemother. 32, 1904-1906). We propose the following mechanism to account for the observed selective toxicity of ethylthioribose. 1) The cytocidal action of ethylthioribose against protozoa is a result of its conversion to ethionine, a well-known cytotoxic agent. 2) This transformation occurs through the pathway which normally converts 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine. 3) Conversion of ethylthioribose to ethionine cannot occur in mammalian cells since these cells cannot phosphorylate methylthioribose (ethylthioribose), a first step in the pathway to methionine (ethionine). To test this hypothesis, [5-3H]ethylthioribose has been synthesized and its metabolism by cell-free extracts of Klebsiella pneumoniae and rat liver was examined. The pathway by which methylthioribose is converted to methionine in K. pneumoniae is well characterized. When supplemented with ATP and L-glutamine, the bacterial extract efficiently converted [5-3H]ethylthioribose to [3H]ethionine. By contrast, ethionine was not produced upon incubation of [5-3H]ethylthioribose, ATP, and L-glutamine with rat liver homogenate. The mammalian cell extract lacks a kinase activity capable of converting ethylthioribose to 1-phospho-5-S-ethyl-5-thio-alpha-D-ribofuranoside, an obligate intermediate in the biosynthesis of ethionine from ethylthioribose in K. pneumoniae. These results support our hypothesis and provide a basis for understanding the apparently selective toxicity of ethylthioribose.

Analogs of 5-methylthioribose, a novel class of antiprotozoal agents.

Since drug resistance and toxicity limit the use of available antiprotozoal agents, it is important that new drugs be developed as soon as possible. In this study, the method by which several protozoa degrade 5'-methylthioadenosine (MTA) was shown to differ from MTA catabolism in human cells. To exploit this metabolic difference, two analogs of methylthioribose (MTR), an MTA catabolite, were synthesized and found to be cytocidal to Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis in vitro. In contrast, these analogs had no effect on cultured mammalian cells. Analogs of MTR represent a potential new class of antiprotozoal drugs.

Biosynthesis of the C29-phytosterol side-chain: evidence that the same stereochemistry at C-25 can originate from different mechanisms

It has been demonstrated that in poriferasterol, biosynthesized in Ochromonas malhamensis, the hydrogen atom originally present at C-24 of the Δ24-precursor is located at C-25.

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Ochromonas malhamensis

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme of the isoprenoid pathway, was found to be predominantly microsomal in Ochromonas malhamensis, a chrysophytic alga. Detection of HMG-CoA reductase requires the presence of 1% bovine serum albumin during cell homogenization, and the activity is stimulated by the presence of Triton X-100. The enzyme has a pH optimum of 8.0 and an absolute requirement for NADPH. When grown in 10 micromolar mevinolin, a competitive inhibitor of HMG-CoA reductase, O. malhamensis shows a 10- to 15-fold increase in HMG-CoA reductase activity (after washing) with little or no effect on cell growth rate. Cultures can be maintained in 10 micromolar mevinolin for months. O. malhamensis produces a large amount (1% dry weight) of poriferasterol, a product of the isoprenoid pathway. The addition of 10 micromolar mevinolin initially blocked poriferasterol biosynthesis by >90%; within 2 days the rate of synthesis returned to normal levels. Immediately after mevinolin was washed from the 2-day culture, there was a transient 2.5-fold increase in the rate of poriferasterol biosynthesis. The rate of poriferasterol biosynthesis and the level of HMG-CoA reductase activity both fell to control levels within hours.

Determination of metabolically active B12 and inactive B12 analog titers in human blood using several microbial reagents and a radiodilution assay.

Metabolically active B12 analogs and inactive B12 analogs were measured in plasma, red blood cells (RBC), and pooled pernicious anemia serum. B12 values by Lactobacillus leichmannii, Escherichia coli, Euglena gracilis, and radioisotope dilution method (RIDA) as assays for total B12 (active analogs + inactive analogs) were compared to Ochromonas malhamensis values as index of only metabolically active B12. B12 values above those with O malhamensis distinguished inactive analogs from active B12. Inactive analogs contribute 85, 97, 135, and 163% above active B12 activity in normal plasma when E gracilis, L leichmannii, RIDA, and E coli, respectively, were used for B12 analysis. RIDA B12 determinations for active B12 in plasma showed that 44% of the B12 measured was still due to inactive analogs when compared to O malhamensis B12 activity. Inactive B12 analogs contributed 21, 151, and 224% above O malhamensis active B12 in RBC when E gracilis, L leichmannii, and E coli, respectively, were used.

Application of biosynthetic 13C enrichment using [1,2‐13C2] acetate as precursor for 13C NMR spectral assignment of the isopropyl methyls of (24β)‐ethylsterols

AbstractThe resonances of C‐26 and C‐27 in the 13C NMR spectra of two (24β)‐ethylsterols, poriferasterol and clionasterol, were unambiguously assigned through a biosynthetic experiment from [1,2‐13C2]acetate in the chrysophyte Ochromonas malhamensis; the conclusion that the hydride migration from C‐24 to C‐25 during the biosynthesis of phytosterols occurs in most cases on the si‐face of the 24‐double bond precursor is experimentally established.

Mechanism of the transmethylation reaction by S-adenosylmethionine: stereochemistry of hydride migration from C-24 to C-25 in the biosynthesis of poriferasterol in the crysophyte Ochromonas malharnensis

Administration of sodium [2-2H3]acetate to the crysophyte Ochromonas malhamensis yielded [2H22]poriferasterol, from which, by chemical degradation, the C(24)-C(29) fragment of the side chain was obtained as (2S,3R)-2-[2H2]methyl[1,1,1,2-2H4]pentan-3-ol. 2H N.m.r. analysis of this deuteriated compound shows that the 1,2-hydride migration from C-24 to C-25 engages the si-face of the Δ24-sterol double-bond, and suggests that S-adenosylmethionine approaches the same double bond from the re- face.

Stereochemistry of hydride migration from C-24 to C-25 in the biosynthesis of priferasterol in Ochromonas malhamensis

2 H-N.m.r. analysis of (2 S, 3 R)-2-([2H2]methyl)-[1,1,1,2-2H4]pentan-3-ol obtained from [2H22]poriferasterol, biosynthesized in the Chrysophyte Ochromonas malhamensis from C2H3CO2Na, shows that the hydride migration from C-24 to C-25 occurs on the si-face of the Δ24-precursor double bond.

Volume Regulation in Poterioochromonas

In Poterioochromonas malhamensis Peterfi (syn. Ochromonas malhamensis Pringsheim) osmotically induced shrinkage is reversed by an accumulation of isofloridoside. Addition of Ca(2+) ions to homogenates from standard volume cells initiates an enzyme system for the activation of isofloridoside-phosphate synthase. This process is stimulated in the presence of Ca(2+) by calmodulin, isolated from the same alga or from bovine brain, and requires the presence of membranes. The stimulation observed when Ca(2+) is added without exogenous calmodulin is inhibited by the calmodulin-binding substance R 24571. These results show that the effect of Ca(2+) is mediated by calmodulin. The Ca(2+)/calmodulin-dependent activation is enhanced when fluoride or molybdate ions are present in the homogenization buffer. This might indicate the involvement of a phosphorylated compound in the activation mechanism.

Synthesis of deuterium labeled cholesterol and steroids and their use for metabolic studies.

A simple method is described for the preparation of [6,7,7(-2)H3] sterols and steroids. The synthesis starts with a Δ(5)-sterol or steroid and involves preparation of the 6-oxo-3α,5α-cyclosteroid, base exchange in the presence of deuterium oxide to introduce two deuteriums at the C-7 position and sodium borodeuteride reduction of the 6-oxo group to introduce the third deuterium atom at C-6. Rearrangement of the [6,7,7(-2)H3]6α-hydroxy-3α,5α-cyclosteroid then gives the desired [6,7,7(-2)H3]-Δ(5) sterol or steroid. [6,7,7(-2)H3]Cholesterol, [6,7,7(-2)H3]pregnenolone and [6,7,7(-2)H3]3β-hydroxyandrost-5-en-17-one were synthesized in this fashion and [6,7,7(-2)H3]progesterone was prepared from the [6,7,7(-2)H3]pregnenolone. Three examples of the use of these deuchromatography-mass spectrometry. The chrysophyte alga,Ochromonas malhamensis, was shown to be capable of introducing an extra methyl or ethyl group at C-24 of the side chain of [6,7,7(-2)H3]cholesterol to yield brassicasterol and poriferasterol, respectively. The ovary of the echinoderm,Asterias rubens, was demonstrated to metabolize [6,7,7(-2)H3]progesterone to yield mainly the 5α-isomers of pregnane-3,20-dione and 3β-hydroxypregnan-20-one. However, the 5β-isomers of these compounds were also detected as minor products for the first time as progesterone metabolites in this animal. Isolated oocytes of the frog,Xenopus laevis, produced a number of metabolites of [6,7,7(-2)H3]progesterone. In this report, two of them were shown to be 17α-hydroxy-pregn-4-en-3,20-dione and 20α-hydroxypregn-4-en-3-one.

Synthesis and Release of Cyclic Adenosine 3′:5′-Monophosphate by Ochromonas malhamensis

The chrysophycean alga, Ochromonas malhamensis Pringsheim, was shown to synthesize cyclic adenosine 3':5'-monophosphate (cAMP) and to release it into the culture medium. Cells contained 3 to 3,000 picomoles per gram fresh weight; medium contained up to 20 times the amount in the cells. Putative [(32)P]cAMP was purified from cultures supplied [(32)P]phosphate. The compound was identified as [(32)P]cAMP by co-chromatography with authentic cAMP through 10 serial steps; by chemical deamination at the same rate as authentic cAMP, to a (32)P compound with the chromatographic behavior of cIMP; and by its conversion through the action of cyclic nucleotide phosphodiesterase to a (32)P compound with the chromatographic behavior of 5'-AMP. A two-step procedure involving chromatography on alumina and on Dowex 50 purified the unlabeled compound from cells or medium sufficiently for it to be assayable by competitive inhibition of binding of [(3)H]cAMP to cAMP-binding protein (Gilman assay) or by stimulation of cAMP-dependent protein kinase. The activity was destroyed by cyclic nucleotide phosphodiesterase with the same kinetics as authentic cAMP, provided that an endogenous inhibitor of the phosphodiesterase was first removed by an additional purification step.

VITAMIN ANALYSIS BY MICROBIAL AND PROTOZOAN ORGANISMS: RESPONSE TO FOOD PRESERVATIVES AND NEUTRALIZATION SALTS

ABSTRACTVitamin analyses using protozoa (Tetrahymena pyriformis, Ochromonas danica and Ochromonas malhamensis) as test organisms were found to he affected by lower concentrations of neutralization salts than were the vitamin analyses which used bacteria and yeast as test organisms. Neutralization salts are formed during vitamin extraction procedures. Growth of protozoa and yeast was affected by the food preservatives‐sodium sorbate, sodium propionate and sodium benzoate. Except for sodium benzoate inhibition of Lactobacillus leichmannii, the growth of bacterial test cultures was not affected by these chemicals. Levels of the neutralization salts and food preservatives in vitamin assays that are required to affect the growth of the test organisms were calculated. These levels were compared to the concentrations of salts formed by accepted vitamin extraction procedures to determine the dilutions of sample extracts necessary to avoid interference. Similar calculations were completed to determine minimal dilutions of foods necessary to avoid the effects of food preservatives on the test organisms. Acids and bases that neutralized to form salts that had the least effect on the growth of the test cultures were determined.

Cryopreservation of Ochromonas in Liquid Nitrogen with Reproducibility of Thiamin Assay

SYNOPSISThe first successful cryopreservation of Ochromonas danica and Ochromonas malhamensis is reported. The freezing method was consistently reproducible for the former, but not for the latter. Ochromonas danica cultures established from frozen material still could be used as test organisms for assay of thiamin. This is the first report of a protozoon retaining its assay property after being frozen to ‐196 C.

VITAMIN ASSAY BY MICROBIAL AND PROTOZOAN ORGANISMS: RESPONSE TO VITAMIN CONCENTRATION, INCUBATION TIME AND ASSAY VESSEL SIZE

ABSTRACTStandard vitamin calibration curves for the microbiological determination of thiamine, riboflavin, vitamin B6, vitamin B1 2, biotin, niacin and pantothenic acid were prepared to determine the minimal and maximal vitamin concentrations that may be assayed by various microorganisms and protozoa, and to determine the incubation periods required for the growth response of the microbial vitamin assays to stabilize. The effect of assay vessel size on growth of Tetrahymena pyriformis was also determined. Vitamin assays using Tetrahymena pyrijkmis were shown to have wider concentration limits than those of bacteria and yeast. Analyses based on Ochromonas danica and Ochromonas malhamensis possessed approximately equal vitamin concentration limits when compared to corresponding bacterial assay methods. Accepted vitamin assay methodology using microorganisms terminate vitamin assays when the growth response has stabilized. This study indicated that some test organisms such as Lactobacillus viridescens, (thiamine), Lactobacillus casei (riboflavin) and Saccharomyces uvarum (vitamin B6) do not achieve a point of stable response, thus requiring a defined incubation period. Tetrahymena pyriformis growth was depressed by use of small diameter assay vessels. Optimum response of this organism requires the use of flasks in which assay media, when dispensed, will have a large surface to volume ratio.

Fine structure of the cell surface and Golgi apparatus of Ochromonas.

Ochromonas danica has an unusually flexible cell surface capable of producing projections of varying sizes and shapes: large projections, 340-360 nm long, and small projections, 50-110 nm long. These projections have been demonstrated by transmission and scanning electron microscopy; some of them may break off into the medium and be the source of extracellular membranes and vesicles reported in the cell-free O. danica growth medium. Ruthernium red stained the acid mucopolysaccharide layer just outside the cell surface as well as small blebs at the cell surface. The Golgi complex of O. danica, Ochromonas malhamensis, Ochromonas sociabilis and Ochromonas sp. produced small coated vesicles which may move toward and fuse with the plasma membrane. The role of the several vesicles is unknown but possible functions are discussed.