Studies of sulfate utilization by algae: 10. Nutritional and enzymatic characterization of chlorella mutants impaired...

Seven mutants of Chlorella pyrenoidosa (Emerson strain 3) impaired for sulfate utilization have been isolated after treatment of the wild-type organism with nitrosoguanidine by replica plating on media containing thiosulfate and l-methionine. These mutants fall into three classes based on their ability to grow on sulfate, accumulate compounds labeled from sulfate-(35)S, and reduce adenosine 3'-phosphate 5'-phosphosulfate-(35)S (PAPS-(35)S) to thiosulfate-(35)S. Mutant Sat(2) (-) cannot grow on sulfate, but it accumulates thiosulfate-(35)S and homocysteic acid-(35)S from sulfate-(35)S in vivo. In addition, extracts of mutant Sat(2) (-) reduce PAPS-(35)S to thiosulfate-(35)S, indicating the possession of enzyme fractions S and A, both of which are required for thiosulfate formation. Mutants Sat(1) (-), Sat(3) (-), Sat(4) (-), Sat(5) (-), and Sat(6) (-) cannot grow on sulfate, and their extracts lack the ability to reduce PAPS-(35)S to thiosulfate-(35)S. Mutant Sat(7) (-)R(1), a probable revertant, can grow on sulfate but still lacks the ability to reduce PAPS-(35)S to thiosulfate-(35)S in vitro. Complementation experiments in vitro show that the block in formation of acid-volatile radioactivity in every case is due to the absence of activity associated with fraction S. All mutants can grow on thiosulfate and all possess the activating enzymes which convert sulfate to PAPS. Through a comparison of nutritional and enzymatic characteristics, the first outlines of a branched and complicated pathway for sulfate reduction in Chlorella are beginning to emerge.