Fermentative Metabolism of Hydrogen-evolving Chlamydomonas moewusii

The anaerobic metabolism of Chlamydomonas moewusii under both light (160 lux) and dark conditions has been examined using manometric and enzymic techniques. During anaerobiosis starch is broken down to glycerol, acetate, ethanol, CO₂, and H₂. The release of CO₂ and H₂ comes to an end when the starch pool is depleted.

There are only slight differences in the ratio of the end products of fermentation between light and dark metabolism. In the light, glycerol production is diminished and H₂ evolution is enhanced, whereas the production rate of all other end products generally does not change.

     

Fermentative Metabolism of Chlamydomonas reinhardtii: II. Role of Plastoquinone

Evidence is presented to substantiate a chloroplastic respiratory pathway in the green alga, Chlamydomonas reinhardtii, whereby reducing equivalents generated during the degradation of starch enter the thylakoidal chain at the plastoquinone site catalyzed by NADH-plastoquinone reductase. In this formulation, the reduced plastoquinone is oxidized either by the photoevolution (photosystem I) of H₂ under anaerobic conditions or by O₂ during dark respiration.     

Fermentative Metabolism of Chlamydomonas reinhardtii: I. Analysis of Fermentative Products from Starch in Dark and Light

The anaerobic starch breakdown into end-products in the green alga Chlamydomonas reinhardtii F-60 has been investigated in the dark and in the light. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) on the fermentation in the light have also been investigated.     

Spectral Dependence of Photoregulation of Inorganic Nitrogen Metabolism in Chlamydomonas reinhardii

The utilization of NO₃⁻ by green algae growing photoautotrophically under air, which are growth conditions close to their more habitual situations in nature, is associated with the excretion of NO₂⁻ and NH₄⁺ to the culture medium. The entire process is promoted by blue light and depends on photosynthetically active radiation for the required reducing equivalents. The stimulation of NO₃⁻ utilization and of its associated NO₂⁻ and NH₄⁺ excretions saturated at very low quantum fluxes of blue light (15 microequivalents per square meter per second) in Chlamydomonas reinhardii cells sparged with CO₂-free air and irradiated with 50 microequivalents per square meter per second background red light. The wavelength dependence data of this stimulation correlated closely with the in situ photoactivation of nitrate reductase and also with the light induced increase in its biosynthesis and/or assembly.

These results indicate that the photoregulation of inorganic N metabolism in C. reinhardii is mainly due to the blue light modulation of nitrate reductase. Although flavins are the most suitable candidates to act as physiological photoreceptors, the wavelength dependence data only show a major peak in the blue region between 400 and 500 nanometers.

     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

DNA polymerases from Chlamydomonas reinhardii. Purification and properties.

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. 'Activated' calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.     

Variability of mitochondrial population in Chlamydomonas reinhardii

Several details have been published cocerning the mitochondrial number and shapes at various stages of the synchronized vegetative and generative cell cycle in Chlamydomonas reinhardii. The present study, based on ultrathin serial sections and threedimensional reconstructions, completes these data. Quantitative analysis of serial micrographs makes it possible to give specific details of mitochondrial volumes in cells at early intermediate stages of the vegetative life cycle. Our investigations clearly show that mitochondria have a relatively wide range of sizes, within certain limits, and vary like the mitochondrial shapes; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. Thus, we present a plastic insight into the dynamically changing micromorphology of the mitochondrial population in Chlamydomonas reinhardii.     

Alternative Acetate Production Pathways in Chlamydomonas reinhardtii during Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.     

The nitrate-reducing enzyme system of Chlamydomonas reinhardii

In Chlamydomonas reinhardii the reduction of nitrate to ammonia occurs in two independent enzymatic steps: 1. the two-electrons reduction of nitrate to nitrite catalyzed by NADH-nitrate reductase, and, 2. the six-electrons reduction of nitrite to ammonia catalyzed by ferredoxin-nitrite reductase. Both enzymes have been purified and characterized, and some of their properties have been studied.     

Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii

Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (−), fix CO₂ at rates between 25 and 50 micromoles per milligram chlorophyll per hour. The upper value is approximately half of the rate of the intact cell.

During storage in the dark on ice, the chloroplast preparation loses 30 to 50% of its CO₂ fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO₂ fixation in the light.

High concentrations of inorganic phosphate (>2 millimolar) inhibit CO₂ fixation. This inhibition is overcome by the addition of glycerate 3-phosphate. It is concluded that chloroplasts from C. reinhardii possess a higher plant type phosphate translocator. With respect to dependency upon light intensity, pH and Mg²⁺ concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO₂ fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer.

     

A deoxyribonuclease from Chlamydomonas reinhardii. 1. Purification and properties.

A deoxyribonuclease has been purified more than 2000-fold from the green algae, Chlamydomonas reinhardii. The enzyme is most active on denatured DNA. Optimum activity is at pH 8.5, in 80 mM Tris-HCl buffer and 2 mM CaCl2. Other divalent cations can replace Ca2+ with varying lower efficiency. EDTA and inorganic phosphate are strongly inhibitory, while ATP and high concentrations of 2-mercaptoethanol are slightly inhibitory. The molecular weight is approximately 35 000, the Stokes radius is 2.7 nm, and the sedimentation coefficient 2.8 S. It is a single polypeptide chain, and the frictional ratio of 1.27 suggests it is only slightly asymetrical. The isoelectric point is 9.5. This enzyme has been termed exonuclease 1.