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Synthesis of Protein and Nucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis¹

^a The Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo, Japan

ABSTRACT

Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating ³²P-orthophosphate into RNA and DNA, ³H-adenosine or ³H-uridine into RNA, and ³H-leucine or ¹⁴C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the glucose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin A₃, streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation.

² Present address: Department of Microbiology, School of Medicine, and the Friday Harbor Laboratories, University of Washington, Seattle, Wash. 98105.

³ Present address: Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo, Japan.

⁴ Present address: Department of Biochemical Sciences, Princeton University, Princeton, N.J. 08540.

⁵ Present address: Department of Agricultural Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.

¹ Presented in part at the XII International Congress of Genetics, Tokyo, Japan, 23 August 1968.