A mutant strain of Chlamydomonas reinhardi lacking ribulose diphosphate carboxylase activity.

Abstract

Mutant strains of the unicellular green alga Chlamydomonas reinhardi which -have impaired photosynthesis are characterized by their inability to fix carbon dioxide in the light at the wild-type rate.l The incapacity of these mutant strains to fix carbon dioxide by photosynthesis can be attributed to one of three possibilities: (1) the loss of a step in the photosynthetic electron transport chain; (2) the loss of the capacity to carry out photosynthetic phosphorylation; or (3) the loss of one of the steps in photosynthetic carbon dioxide fixation associated with the reductive pentose phosphate cycle. The mutant strains of C. reinhardi described to date2-4 fall into the first category mentioned above. In this paper we shall describe ac-20, a mutant strain which lacks the capacity for photosynthetic carbon dioxide fixation as a consequence of the loss of RuDP carboxylase activity. Organisms and the Methods.-The organisms used in the experiments described below were the wild-type strain of C. reinhardi, 137c, and the mutant strain ac-20, derived from wild type by ultraviolet irradiation followed by a screening test'; for carbon dioxide fixation. Cells, in the logarithmic phase of growth, were harvested from shake cultures grown at 25?C in high salt minimal medium' supplemented with 0.2% sodium acetate. Light (2500 lux) was provided by daylight fluorescent lamps. Carbon dioxide fixation by whole cells was measured as previously described.7 The light intensity was 60,000 lux. The water-soluble products of carbon dioxide fixation by whole cells in the light were also examined. A cell suspension in minimal medium (30 ml, 10 mg/ml wet weight) was placed in a lollipop and illuminated with 20,000 lux. Temperature was maintained at 25?C. The suspension was aerated, and after 10 min C14-labeled sodium bicarbonate (8 ,moles, 50 /c/imole) was introduced into the lollipop. Sixty sec later the reaction was terminated by emptying the contents of the lollipop into a hot methanol-chloroform mixture (12:5 v/v). The water-soluble products were then extracted according to the method of Bieleski and Young.' The products were separated on a Dowex-l chloride column (1 X 90 cm) using two stages of linear HC1 gradient elution. The gradient was as follows: tubes 1-35, water; tubes 36-155, a linear gradient from 0.028 N to 0.041 N HC1; tubes 156-265, a linear gradient from 0.070 N to 0.130 N HC1. Aliquots of 100 lambda from each tube were plated on planchets and counted. Twelve peaks were observed. Chloroplast fragments were prepared according to the method of Levine and Volkmann.K Crude extracts for enzyme assays were obtained from cells which had been washed once in 0.02 M Tris buffer, pH 7.5, and then resuspended in 5 ml of the same buffer. The cells were disrupted by sonic oscillation at 0?C for 21/2 min using a Mullard 20-kc ultrasonic disintegrator. The disrupted cell preparations were then centrifuged at 20,000 X g for 20 minl at 0?C. The green supernatant was used as the crude extract. The activity of the photosynthetic electron transport chain was measured by assaying the rate of TPN photoreduction by chloroplast fragments using the method described by Levine and Smillie.2