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We have examed the induction of carbonic anhydrase activity in Chlanydomona reiahardtli and have Identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photonutotrophically on 5% C(0, but its synthesis was induced wader low concentrations of (20 (air levels of (20 The photosynthetic characteristics of the green alga Chlamydomonas reinhardtii are dependent upon the C02 concentration experienced by the alga during growth (1, 2). Cells grown at air levels of C02 (0.03%) can utilize low C02 concentrations much more efficiently than cells grown at high levels of C02 (3-5%). Since the C02 concentration during growth has no effect on either the mechanism of photosynthetic C02 fixation or the Km (C02) of the principal C02 fixing enzyme, RuBP4 carboxylase (2), aikgrown algae must employ another method for increasing the efficiency of C02 utilization. Recent work has demonstrated that the appearance of both CA activity and a mechanism for ' C(02(11, 13, 16). While CA and the C, transport system increase following the transfer of C. reinhardtii from high to low levels of C02 and appear to be required for the maintenance of C02 fixation in an environment deficient in CQ, the exact manner in which these activities coordinate Ci accumulation is not fully known.In this study, we have examined the adaptation of C. rein/ardtii to air levels of C02. The photosynthetic capacity of the organism at low C02 concentrations rapidly increases after a shift from growth on high to low C02 and, S h following the transfer, the alga displays photosynthetic characteristics similar to those observed in air-grown cultures. We have compared the kinetic parameters of C02 fixation during adaptation with the induction of CA and have localized and identified the major species responsible for CA activity using both biochemical and immunological procedures. MATERIALS AND METHODSChlamydomonas reinhardtii 2137 mt+ (obtained from Dr. M. Spalding, Michigan State University) and the cell wall-less mutant, CW-15, were cultured axenically in the minimal medium described by Spalding et al. (13) at 28C and a light intensity of 300 ,#E m-2 s-' (400-700 nm). Cultures were vigorously shaken and bubbled with either 5% C02 in air or with air alone. All experiments were performed with cells in early to midphase exponential growth.For the determination of K112 (C02) and the maximal rate of photosynthesis, P,,,r, algae grown under the appropriate conditions were harvested by centrifugation (4,000g), resuspended in C202-free, 20 mm 3(N-morpholino)propanesulfonic acid buffer (pH 7.2), and the rates of 02 evolution at varying HC03-concentrations were measured at saturating light intensity and 25(C with a Clark type 02 electrode. CA activity in cell pressates, prepared as described below, was determined electrometrically and exrs in Wilbur-Anderson units (WA) (9, 18).For studying the induction and purification of CA we used cultures ofthe C. reinhardtii mutant, CW-1S. This mutant lacks a cell wall,...

A Chiamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO2 concentrations but can grow on elevated CO2. In a test cross, the high C02-requirement for growth showed a 2:2 segregation. This mutant, designated CIA-5, had a phenotype similar to previously identified mutants that were defective in some aspect of CO2 accumulation. Unlike previously isolated mutants, CIA-5 did not have detectable levels of the periplasmic carbonic anhydrase, an inducible protein that participates in the acquisition of CO2 by C. reinhardtii. CIA-5 also did not accumulate inorganic carbon to levels higher than could be accounted for by diffusion. This mutant strain did not synthesize any of the four polypeptides preferentially made by wild type C. reinhardtii when switched from an environment containing elevated CO2 levels to an environment low in CO2. It is concluded that this mutant fails to induce the CO2 concentrating system and is incapable of adapting to low CO2 conditions. Chlamydomonas reinhardtii, like other unicellular green algae, has the capacity to adapt to varying CO2 concentrations in the environment (1-3, 5). When grown on elevated levels of CO2 (5% (v/v) in air), C. reinhardtii has a relatively low affinity for C12 and exhibits high rates of photorespiration when placed in a low CO2/high O2 environment (2,16,19,26). However, if C. reinhardtii remains exposed to low levels of CO2 (ambient CO2 levels), it adapts to these conditions by inducing a CO2 concentrating mechanism (2). This CO2 concentrating mechanism is thought to increase the CO2 at the

Isolated intact chloroplasts from wall-less mutants of Chlamydomoias reinhardtii accumulate inorganic carbon (C1) from the medium provided the cells had been adapted to low CO2 photoautotrophic growth conditions. Chloroplasts from cultures grown on high (5%) CO2 or photoheterotrophically with acetate did not accumulate inorganic carbon. Chloroplast C, accumulation from low CO2 grown cells was light dependent and was inhibited by uncouplers and inhibitors of electron transport. In a model for C, accumulation by Chiamydomonas, it is proposed that CO2 diffuses into the cell and Ci accumulation occurs in the chloroplast.

Abstract. The unicellular green alga Chlamydomonas reinhardtii responds to sulfate deprivation by producing an arylsulfatase (Lien, T., and O. Schreiner. 1975. Biochim. Biophys. Acta. 384:168-179; Schreiner, O., 1975. Biochim. Biophys. Acta. 384:180-193) and by developing the capacity to transport sulfate more rapidly (our unpublished data). The arylsulfatase activity, detectable 3 h after the transfer of the cells to low sulfate medium (~<10 IxM sulfate), is a periplasmic protein released into the culture medium by cw15, a cell wall-less mutant of C. reinhardtii. We have purified the derepressible arylsulfatase to homogeneity and have raised monospecific antibodies to it. The protein monomer (67.6 kD) associates into a dimer, and the enzyme activity shows an alkaline pH optimum and a Km of 0.3 mM for p-nitrophenylsulfate. Studies focused on arylsulfatase biosynthesis demonstrate that it is glycosylated and synthesized as a higher molecular mass precursor. The mature protein contains complex N-linked oligosaccharides and the primary translation product has an apparent molecular mass ",,5 kD larger than the deglycosylated monomer. Since translatable RNA encoding the arylsulfatase can only be detected in cells after sulfate starvation, it is likely that accumulation of the enzyme is regulated at the level of transcription, although posttranscriptional processes may also be involved.

Photoproduction of H2 by green algae utilizes electrons originating from the photosynthetic oxidation of water and does not require metabolic intermediates. However, algal hydrogenases are extremely sensitive to O(2), which limits their usefulness in future commercial H2-production systems. We designed an experimental technique for the selection of O2-tolerant, H2-producing variants of Chlamydomonas reinhardtii based on the ability of wild-type cells to survive a short (20 min) exposure to metronidazole in the presence of controlled concentrations of O2. The number of survivors depends on the metronidazole concentration, light intensity, preinduction of the hydrogenase, and the presence or absence of O2. Finally, we demonstrate that some of the selected survivors in fact exhibit H2-production capacity that is less sensitive to O2 than the original wild-type population.

Photoproduction of H2 by green algae utilizes electrons originating from the photosynthetic oxidation of water and does not require metabolic intermediates. However, algal hydrogenases are extremely sensitive to O2, which limits their usefulness in future commercial H2-production systems. We designed an experimental technique for the selection of Ortolerant, H2-producing variants of Chlamydomonas reinhardtii based on the ability of wild-type cells to survive a short (20 min) exposure to metronidazole in the presence of controlled concentrations of O2, The number of survivors depends on the metronidazole concentration, light intensity, preinduction of the hydrogenase, and the presence or absence of O 2 , Finally, we demonstrate that some of the selected survivors in fact exhibit H2-production capacity that is less sensitive to O2 than the original wild-type population.Index Entries: Hydrogen; green algae; Chlamydomonas; oxygen; metronidazole.Abbreviations: ATP, adenosine triphosphate; cw15, cell wall-less strain of Chlamydomonas reinhardtii; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; Fd, ferredoxin; Iso, inhibitor concentration that decreases the rate of an enzymatic reaction to 50% of the rate measured in the absence of the inhibitor; MNZ, metronidazole, [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole]; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP; V o , initial rate of an enzymatic reaction; WT, wild-type.

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